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(Received for publication, August 25,
1995; and in revised form, January 10, 1996) From the
Using four cultured cell models representing liver,
keratinocyte, and osteoblast, we have demonstrated that the vitamin D
analog, 22-oxacalcitriol is degraded into a variety of hydroxylated and
side chain truncated metabolites. Four of these metabolic products have
been rigorously identified by high pressure liquid chromatography,
diode array spectrophotometry, and gas chromatography-mass spectrometry
analysis as 24-hydroxylated and 26-hydroxylated derivatives as well as
the cleaved molecules, hexanor-1
It is now firmly established that the hormonal form of vitamin
D, 1 OCT binds poorly
to the vitamin D binding protein (DBP) and is transported in the plasma
bound by lipoproteins (chylomicrons and low density lipoprotein) in
vivo(9) , and this leads to an unusual distribution
pattern with a degree of concentration of the vitamin D analog in
parathyroid tissue(10) . In contrast, little is known about OCT
metabolism except that it appears to be excreted as a glucuronide
conjugate in the bile, possibly a derivative of a truncated version of
OCT(10) . Furthermore, there have been suggestions that a
metabolite with a truncated side chain may be formed in bovine
parathyroid cell cultures in vitro(11) , although
details of this have yet to be published. In these studies we set
out to provide convincing physicochemical identification for
metabolites generated in a variety of cultured cell models, namely the
human hepatoma lines HepG2 and Hep3B(12) , the rat osteosarcoma
cell line UMR-106(13) , and the human keratinocyte cell lines
HPK1A and HPK1A-ras(14, 15) . Because these
cell models mimic vitamin D metabolism found in vivo, we
expected to observe the same metabolites found and in some cases
tentatively identified by others(10, 11) . In
addition, our objective was to study the rate of OCT metabolism in
various cell lines in order to gauge the involvement of vitamin
D-inducible catabolic pathways as compared with more general
metabolizing systems. Our results support the concept that OCT is
subject to extensive metabolism in a variety of tissues that leads to
side chain truncated forms excreted in the bile.
Figure 1:
HPLC of extract of HPK1A-ras cells incubated with 10 µM OCT for 48 h. Peaks
showing the vitamin D chromophore are numbered 1-7. A shows the no cell control experiment. B shows the
extract from HPK1A-ras cells. the HPLC conditions were
Zorbax-SIL (3 µ; 6.2
Figure 2:
A,
mass spectrum of pertrimethylsilylated peak 2. B, mass
spectrum of pertrimethylsilylated oxime derivative of peak
2.
Treatment of peak 2 with sodium
borodeuteride gave two product peaks, one of which co-migrated with
peak 4 (peak 2 (untreated) = 10.77 min; peak 2 treated with
NaBD
Figure 3:
A, mass
spectrum of pertrimethylsilylated peak 4. B, mass spectrum of
pertrimethylsilylated derivative of the sodium borodeuteride reduction
product of peak 2.
Final
proof of the identity of peak 2 as the 20-ketone came from formation of
an oxime derivative. When the oxime was further derivatized with N-trimethylsilylimidazole, it gave a pertrimethylsilylated
derivative with a molecular ion of m/z 503 (Fig. 2B). Major fragment at 413
(M
Figure 4:
A, mass spectrum of pertrimethylsilylated
peak 6. B, mass spectrum of pertrimethylsilylated derivative
of the n-butyl boronate ester of peak
6.
The n-butylboronate derivative of peak 6 was
formed, again suggesting vicinal hydroxyls at C-24 and C-25. The
pertrimethylsilylated n-butyl boronate of peak 6 gave the mass spectrum (Fig. 4B) with the predicted molecular ion of m/z 644 and a fragmentation pattern particularly m/z = 169 due to cleavage of the oxygen
O-22-C-23 bond consistent with a 24-hydroxylated precursor.
Finally, peak 6 was shown to co-chromatograph with chemically
synthesized 24R-OH-OCT on HPLC. We conclude on the basis of
extensive evidence that peak 6 is 24R-hydroxy-OCT.
Figure 5:
A, mass
spectrum of pertrimethylsilylated peak 7. B, mass spectrum of
pertrimethylsilylated derivative of the n-butyl boronate ester
of peak 7. C, mass spectrum of pertrimethylsilylated
derivative of the sodium periodate cleavage product of peak
7.
On GC-MS, the periodate cleavage product of peak 7 gave no
discernible molecular ion, but the fragmentation pattern featured large
fragments at m/z 456 (M
Figure 6:
Time
course of OCT metabolism in HPK1A-ras cells.
Figure 7:
HPLC of extract of other cell lines
incubated with 10 µM OCT for 48 h. A, HPK1A
cells. B, UMR-106 cells. The HPLC conditions are as described
in the Fig. 1legend.
When we compared metabolism of OCT at
the same time, under the same experimental conditions (Fig. 8),
we confirmed that OCT had virtually disappeared at the end of the 48-h
period in HPK1A-ras cells, whereas considerable amounts
remained in plates containing UMR-106 and HPK1A cells. The major
metabolite found in cell cultures of UMR-106 and HPK1A was the
24-hydroxy-OCT (peak 6), whereas the truncated metabolites, peaks 4 and
2, appeared to be more important as the degree of metabolism increased (e.g. in HPK1A-ras). These data are consistent with
the scheme shown in Fig. 9with monohydroxylated versions of OCT
acting as intermediates to the truncated metabolites, peaks 4 and 2.
Figure 8:
Amounts of metabolites formed from OCT in
various cultured cell lines. The results are expressed as the total
amount of product formed (and thus isolated) in µg/48 h. The amount
of OCT remaining in the cultures was 1.4 µg for HPK1A-ras,
26 µg for HPK1A, and 28.5 µg for UMR106 and was not determined
for Hep G2.
Figure 9:
Proposed pathway of OCT metabolism in
cultured vitamin D target cells in
vitro.
Figure 10:
Biological activity of the metabolites of
OCT (relative to 1
Figure 11:
Structures and biological activity data
for chemically synthesized metabolites of OCT. The data are summarized
for both stereochemical versions of each
metabolite.
The measurements of the DBP-binding affinity (Fig. 10B) confirmed earlier observations (4, 9) that OCT showed a value 2 orders of magnitude
lower than 1 In
the growth response study (Fig. 10C),
1 We have described here the extensive metabolism of the
vitamin D analog, 22-oxacalcitriol in a variety of cultured cell lines,
including one hepatoma cell line and three vitamin D-target cell lines
previously shown to catabolize 1 The finding of both intact and
truncated versions of OCT in cell extracts evokes the obvious question
of how is OCT cleaved? Our venture into the metabolic fate of an oxygen
containing molecule was not without precedent. In a previous study, we
examined the metabolic fate of 24-oxa-1 Only in the case of the keratinocyte
cell lines (HPK1A-ras and HPK1A) did we find appreciable
quantities of the 20-oxo-truncated derivative of OCT. It is possible
that the pattern of OCT metabolism that we observed, including the
formation of the 20-oxo derivative, resulted from the induction of the
catabolic enzymes caused by the high substrate concentrations that we
employed. Interestingly, we observed the same pattern of metabolites
including the 20-hydroxy and 20-oxo-truncated derivatives when
[2 The biological
activities of the metabolites identified here (where possible together
with their epimeric forms; see Fig. 11) provides evidence that
the in vivo properties of OCT can probably be ascribed to the
parent compound and not to its principal products. In all cases,
metabolites of OCT were inferior to OCT (and
1 In the work described
here, we observed OCT metabolism in both vitamin D target cells,
namely, bone and keratinocyte, as well as a tissue involved in the
activation of vitamin D, namely liver. It has been previously shown
that like 1 Lastly, our finding of truncated versions of
OCT would seem to open the door to study the origin of the conjugates
found in bile in vivo following OCT administration to
rats(10) . The free 20-oxo or 20-hydroxy compounds identified
here would likely be transported to the liver prior to conjugation to
glucuronic acid in the hepatocyte, but this remains to be proven.
Volume 271,
Number 15,
Issue of April 12, 1996 pp. 8700-8708
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,20-dihydroxyvitamin D
and hexanor-20-oxo-1-hydroxyvitamin D.
Comparison with chemically synthesized standards has revealed the
stereochemistry of the biological products. Although differences exist
in the amounts of products formed with the different cell types, it is
apparent that 22-oxacalcitriol is subject to metabolism by both vitamin
D-inducible and noninducible enzymes. Time course studies suggest that
the truncated 20-alcohol is derived from a side chain hydroxylated
molecule via a hemiacetal intermediate and the 20-oxo derivative is
likely formed from the 20-alcohol. Biological activity measurements of
the metabolites identified in our studies are consistent with the view
that these are catabolites and that the biological activity of
22-oxacalcitriol is due to the parent compound. These results are also
consistent with recent findings of others that the biliary excretory
form of 22-oxacalcitriol is a glucuronide ester of the truncated
20-alcohol.
,25-dihydroxyvitamin D (1,
25-(OH)
D or calcitriol) (
)has potent
cell differentiating/anti-proliferative activities in addition to its
role in calcium homeostasis(1) . This has led researchers in
both universities and the pharmaceutical industry to search for
so-called ``noncalcemic'' vitamin D analogs with accentuated
differentiating/anti-proliferative properties and reduced ability to
cause hypercalcemia(2, 3) . 22-Oxacalcitriol (OCT) ()was an early analog developed for this purpose and
contains a 22-oxygen atom that replaces the 22-carbon of calcitriol.
OCT binds to the chicken vitamin D receptor (VDR) with an approximately
8 times lower affinity than
1,25-(OH)D(4) . However, OCT is 10
times more effective than 1,25-(OH)D in suppressing cell growth and inducing differentiation of the
mouse myelocytic leukemic cell, WEHI-3, in vitro(5) .
OCT also possesses an enhanced in vivo immunomodulatory
potency in mice that is 50 times higher than that of
1,25-(OH)D(6) . In contrast, OCT
has reduced calcemic activity in vivo (- that of 1,
25-(OH)D), both in terms of mobilizing calcium
from bone and in stimulating intestinal calcium transport in vitamin
D-deficient and normal rats(7, 8) . The mechanisms
responsible for these differences in biological activity remain
unclear, but factors such as cellular uptake and intracellular
metabolism could contribute to these differences.
Materials
1,25-(OH)D was a generous gift
from Dr. M. Uskokovic of Hoffmann-LaRoche. OCT was synthesized by the
method of Murayama et al.(7) . Putative metabolites of
OCT, including 22,23,
24,25,26,27-hexanor-1,20-(OH)D and
22,23,24,25,26,27-hexanor-20-oxo-1-hydroxyvitamin D as
well as 24- and 26-hydroxylated derivatives, were synthesized as
recently described(16) . 25-OH-D and
[23,24(n)-H]25-OH-D (3.33 TBq/mmol)
were obtained from Duphar Co. (Weesp, Holland) and Amersham Corp.,
respectively. Cell culture media and fine chemicals were purchased from
Life Technologies, Inc. Fetal calf serum was from Flow Laboratories
(Costa Mesa, CA). Derivatizing agents and solvents were purchased as
follows: sodium periodate (BDH Chemicals Ltd, Poole, UK); sodium
borodeuteride (Merck, Sharp, & Dohme, Montreal, PQ, Canada); N-trimethylsilylimidazole (Pierce); and HPLC grade organic
solvents (Caledon, Georgetown, ON, Canada). All other reagents were
analytical reagent grade.Cell Culture
The cell lines UMR106 and HepG2 were cultured as described
previously(12, 13) . Immortalized (HPK1A) and
transformed (HPK1A-ras) cell lines were established from
normal human keratinocytes by stable transfection with human papilloma
virus type 16 followed by transformation with the activated H-ras oncogene, respectively(14, 17) . HPK1A and
HPK1A-ras cell lines were cultured as described
previously(15) .Generation of OCT Metabolites
As described previously(15) , 150-mm culture dishes
containing monolayers (HPK1A, HPK1A-ras, UMR106, or HepG2)
cultured to confluence in the presence of fetal calf serum were washed
with phosphate-buffered saline and then incubated in Dulbecco's
minimal essential medium containing 1% bovine serum albumin in the
presence of 41.8 µg of OCT (10 µM) for 48 h at 37
°C. Except in the time course experiments described below, cells
were not treated with 1,25-(OH)D to induce
catabolic enzymes. No cell or dead cell controls consisted of 10 ml of
medium or microwave-treated cells incubated with OCT under the same
conditions for the same length of time. Medium and cells were removed
for analysis of OCT metabolites, whereas representative plates of cells
were used for determination of cell number using a model ZM automatic
cell counter. In the time course study, HPK1A-ras cells were
cultured to late log phase in Dulbecco's minimal essential medium
containing 10% fetal calf serum and treated with 10 nM 1,25-(OH)D for 16 h in order to
maximally induce catabolic enzymes from the beginning. Monolayers were
then washed with phosphate-buffered saline containing 2% bovine serum
albumin in order to remove residual
1,25-(OH)D, and then the medium was
changed and the cells were incubated with 41.8 µg of OCT (10
µM) in 10 ml/dish Dulbecco's minimal essential
medium containing 1% bovine serum albumin for 0, 1, 3, 6, 12, and 24 h. Lipid Extraction
Lipid extraction was performed according to the method of
Bligh and Dyer (18) as modified by Makin et
al.(19) . The organic layer, containing unchanged OCT and
lipid-soluble metabolites, was evaporated to dryness, and the residue
redissolved in hexane/isopropanol/methanol (HIM), (91:7:2, v/v/v) for
straight phase HPLC.High Performance Liquid Chromatography
Straight phase HPLC was carried out using a model 590 pump, a
U6K manual injector, and a model 990 photodiode array detector (all
from Waters Associates, Milford, MA). This scanning spectrophotometer
was used during metabolite purification to identify peaks possessing UV
characteristics of the vitamin D cis-triene system (UV
= 265 nm, UV
= 228 nm, UV
= 1.75).
Separations were achieved using Zorbax SIL (6.2 
80 mm) or
Zorbax CN (4.6 250 mm) columns (Dupont NEN). Mobile phases used
for OCT metabolites included mixtures of HIM ranging from 91:7:2 to
94:5:1. Concentrations of stock solutions of OCT and its metabolites
were assessed spectrophotometrically using a molar extinction
coefficient, 
= 18,300.Purification of Metabolites
The lipid residue from the extraction of three 150-mm culture
dishes containing HPK1A-ras (or HPK1A) cells was redissolved
in 1.2 ml of HIM 91:7:2. Initial chromatography was performed on Zorbax
SIL using HIM 91:7:2 at flow rate 1.0 ml/min. Six 200-µl aliquots
of the extract were chromatographed, and peaks possessing the
characteristic chromophore of vitamin D were collected and pooled.
These fractions were subjected to further chromatography on Zorbax CN
and Zorbax SIL. At this point, metabolites were pure enough for
chemical derivatization and/or mass spectrometry.Derivatization Reactions
Periodate oxidation and borohydride reduction of metabolites
of OCT were carried out according to published
methods(13, 20) .Gas Chromatography-Mass Spectrometry
Purified metabolites were derivatized to pertrimethylsilyl
ethers and then analyzed by GC-MS as described previously(20) .
Injection of metabolites of OCT into the high temperature injection
zone of the GC causes B ring closure producing pyro- and
isopyroisomers. Mass spectra were obtained only from the pyroisomer,
and, for simplicity, in discussion of fragmentation and in figures
illustrating spectra obtained, the uncyclized metabolite structure is
used rather than that of the correct pyroisomer. Mass spectra were
obtained by averaging each peak and subtracting the background.Biological Properties of OCT and Its Metabolites
VDR Binding Assay
The binding affinities of OCT
and its metabolites for the calf thymus vitamin D receptor were tested
using a 1,25-(OH)D assay kit purchased
from INCSTAR (Stillwater, MN). Calf thymus VDR was incubated at 20
°C for 1 h with various concentrations of
1,25-(OH)D (1.25-80 pg/tube), OCT
(1.25-80 pg/tube), or its metabolites (2.5 pg/tube to 205
ng/tube). After the incubation period, 15,000 dpm of
[H]1,25-(OH)D was
added and incubated for 1 h at 20 °C. Bound and free forms of
[H]1,25-(OH)D were
separated by addition of a dextran/charcoal suspension and
centrifugation. Radioactivity was measured with an Aloka LSC-90.DBP Binding Assay
The binding affinities of OCT
and its metabolites for the DBP were tested using plasma from a vitamin
D-deficient rat. The displacement of
[H]25-OH-D from 1:70,000 diluted
vitamin D-deficient rat plasma in 3.5 mM barbiturate buffer
(pH 8.6) containing 0.13 M NaCl and 0.1% ovalbumin was
measured following the addition of 25-OH-D (12.5 pg/tube
-3.2 ng/tube), 1,25-(OH)D (1.6-820 ng/tube), or OCT or its metabolites (51 ng/tube to
52.4 µg/tube) as described previously(21) . After
incubation for 1 h at 4 °C, bound and free forms of
[H]25-OH-D were separated by the
addition of a dextran/charcoal suspension and centrifugation. The
supernatants containing bound [H]25-OH-D were transferred into scintillation vials, and radioactivity was
measured in a scintillation counter.Assessment of Cell Growth of
HL-60(22
Human promyelocytic leukemic cells (HL-60
cells) were kindly provided by Dr. M. Inaba (Osaka City University,
Medical School). Cells were cultured at 37 °C in RPMI 1640 medium
(Nissui Pharmaceutical, Japan) supplemented with 10% heat-inactivated
fetal calf serum and 60 µg/ml of kanamycin in a humidified
atmosphere of 5% CO in air. Under these conditions the
doubling time of HL-60 cells was 24 h. Vitamin D treatment involved
seeding HL-60 cells at 1 10
cells/ml in growth
medium and culturing for 72 h in the presence of from 10
M to 10
M of
1,25-(OH)D, OCT, or its metabolites, each
dissolved in ethanol. Control cultures were treated with ethanol
vehicle at 0.1% (v/v). After the incubation period, cells were
harvested, and the cell number was determined using a hemocytometer.
Cell viability was determined by trypan blue exclusion. The number of
cells counted from triplicate experiments was expressed as a percentage
of the control. The results are expressed as the mean of triplicate
counts ± standard error.
Metabolism of OCT in the Human Keratinocyte Cell Line,
HPK1A-ras
In an attempt to establish the full extent of metabolism of
OCT, we began our studies by incubating this analog with the cell line
HPK1A-ras, previously shown to have very high catabolic
activity toward 1,25-(OH)D(15) .
When we incubated 10 µM OCT for 48 h, we obtained
extensive metabolism such that on HPLC seven metabolite peaks
possessing the vitamin D chromophore were discernible. These are
labeled peaks 1-7 in Fig. 1, where peak 3 is the
substrate, OCT, and the rest are metabolic products. Little substrate
was left at the end of the 48-h period, whereas no metabolites were
formed in no cell or dead cell (microwave-treated) control plates
incubated with OCT (Fig. 1A). All peaks 1-7 were
purified by extensive rechromatography on Zorbax-CN and then again on
Zorbax-SIL in order to identify them by GC-MS and chemical
derivatization. In the case of peak 2, we made a sodium borodeuteride
reduction product and an oxime as well as the pertrimethylsilylated
derivative. In the case of peaks 4, 6, and 7, they were subjected to
GC-MS as the pertrimethylsilylated derivatives or, in the case of peaks
6 and 7, as their n-butyl boronate ester/pertrimethylsilylated
derivatives.
80 mm); HIM 91:7:2, 1.0
ml/min.
Identification of the Major Metabolites of OCT
We have summarized in Table 1and detailed below the
evidence that was obtained for the successful identification of the
major metabolites. Peak 3 was identified as unchanged substrate. We
obtained limited information regarding peaks 1 and 5, and these
metabolites remain unidentified at this time.
Peak 2 (22,23,24,25,26,27-Hexanor-20-oxo-1
This peak ran at 10.72 min, slightly ahead of the
substrate OCT (RT = 11.41 min) on Zorbax-SIL
chromatography. Peak 2 was significantly retarded relative to OCT on
Zorbax-CN chromatography (see Table 1), suggesting the presence
of a C=O group in the molecule. The pertrimethylsilylated
derivative of peak 2 showed a molecular ion at m/z 474 on GC-MS (Fig. 2A), suggesting truncation of
the side chain between the 22-oxa-group and the C-23 and retention of
the 22-oxa group as a 20-ketone functionality with the subsequent
addition of only two TMS groups. The remainder of the mass spectrum
with major ions at m/z 384(474 - 90), 343 (loss
of a fragment containing carbons C-2,3,4), 294(474 - 90 -
90), and 217 (loss of a fragment containing carbons C-1,2,3) was
consistent with this interpretation.-hydroxy vitamin
D
product 1 = 12.88 min and product 2 =
14.94 min; peak 4 (untreated) = 14.91 min using Zorbax-SIL, HIM,
91:7:2 1 ml/min). When the NaBD reduction product of peak 2
was further derivatized with N-trimethylsilylimidazole, the
pertrimethylsilylated product gave a molecular ion of m/z 549 (Fig. 3B) corresponding to the addition of one
deuterium atom to the original molecule and fragments at m/z 459(549 - 90), 418 (549 - 131 due to
loss of a fragment containing carbons C-2,3,4), 369(549 - 90
- 90), and a base fragment of m/z 118
corresponding to cleavage of the C-17-C-20 bond. This spectrum is
very similar to that of peak 4 and is consistent with the reduction of
the 20-ketone to a mixture of monodeuterated 20-alcohols.
-90), 382 (M-OCH-90),
370 (M-131 due to loss of a fragment containing
carbons C-2,3,4), and 292
(M-OCH-90-90) are all consistent
with a 20-oxime derivative. We conclude from HPLC, GC-MS, borohydride
reduction, and oxime formation that peak 2 is the truncated 20-ketone.Peak 4
(22,23,24,25,26,27-Hexanor-1
Peak 4 was more strongly retained than OCT at
14.91 min on Zorbax-SIL chromatography. Peak 4 co-migrated with
synthetic hexanor-1,20-dihydroxyvitamin
D),20S-dihydroxyvitamin D on both
Zorbax-SIL and Zorbax-CN. GC-MS of the pertrimethylsilylated derivative
of peak 4 gave the mass spectrum shown in Fig. 3A and
can be seen to be homologous to the monodeuterated peak 2 (Fig. 3B). Pertrimethylsilylated peak 4 possessed a
molecular ion of m/z 548 corresponding to a C
sterol with three TMSOH groups. The fragmentation pattern shows
the expected features with m/z 458 (M - 90), 368 (M - 90 - 90), and
417 (M - 131 due to loss of a fragment
containing carbons C-2,3,4), but in addition, a fragment at m/z 117 corresponding a C fragment from
C-20 and C-21 bearing a TMS group. We conclude peak 4 is the truncated
20-alcohol derivative of OCT.Peak 6 (24-Hydroxy-OCT)
This peak was the major
product of all the cell types tested, and because all are known to be
rich sources of 24-hydroxylase activity, it seemed logical that peak 6
would prove to be the 24-hydroxy derivative of OCT. A moderately polar
peak on Zorbax-SIL, this peak was sensitive to sodium metaperiodate
treatment, suggesting the presence of vicinal hydroxyl functions at
C-24 and C-25. On GC-MS (Fig. 4A) the
pertrimethylsilylated derivative of peak 6 possessed a molecular ion of m/z 722 with major fragments at 632 (M - 90) and 542 (M - 90 - 90) as
well as m/z 591 (M - 131 due
to loss of a fragment containing carbons C-2,3,4) and m/z 459, 369, and 279 found in the original substrate, suggesting no
alteration of the ring structure. The base peak at m/z 131 is indicative of retention of the C-25 hydroxyl function
intact.
Peak 7 ((25R)-26-Hydroxy-OCT)
This peak was the
most polar metabolite obtained with a retention time of 23.99 min on
Zorbax-SIL and 16.73 min on Zorbax-CN (Table 1). Like peak 6, it
proved to be sensitive to sodium metaperiodate, giving a product with a
much reduced retention time (12.13 min versus OCT =
11.35 min). On GC-MS, the pertrimethylsilylated derivative of peak 7
gave a molecular ion of m/z 722, suggesting a
monohydroxylated derivative of OCT. The mass spectrum (Fig. 5A) featured many of the same ions found in the
spectrum of peak 6 except that there was a large fragment at m/z 103 (corresponding to loss of CHOTMS)
instead of m/z 131 found in the case of
24-hydroxy-OCT. This fragment is indicative of a CHOTMS
presumably vicinal to the existing C-25 hydroxy group at C-26 (C-27).
- 90)
and 366 (M - 90 - 90) and m/z 415 (M - 131 due to loss of a fragment
containing carbons C-2,3,4), data consistent with the molecular ion
being extrapolated to be m/z 546 (Fig. 5C). This is consistent with a molecule truncated
between C-25 and C-26 (C-27) and containing a C-25 ketone. Further
confirmation of the peak 7 being a molecule with vicinal hydroxyls came
from formation of an n-butylboronate derivative. As with peak
6 the pertrimethylsilylated version of this was subjected to GC-MS and
gave a molecular ion of m/z 644 (Fig. 5B). In addition to the distinctive fragment at m/z 169 found with the n-butylboronate,
pertrimethylsilylated derivative of 24-hydroxy-OCT, we observed a
distinctive fragment at m/z 141 due to cleavage of
the C-24-C-25 bond and loss of a fragment containing the C-25,
C-26, and C-27 butylboronate ester group. Finally, peak 7 was shown to
co-chromatograph with chemically synthesized (25R)-26-OH-OCT
on HPLC. We conclude from these data that peak 7 must be
(25R)-26-hydroxy-OCT.Comparison of Biological Products to Chemically
Synthesized Standard Compounds
As a result of preliminary in vitro studies, both
possible epimers of each biologically generated metabolite were
synthesized chemically. Table 2shows the results of HPLC
co-migration studies of biological and chemically synthesized
metabolites. In each case the epimeric mixture could be resolved on
HPLC, and the biological product was found to be exclusively one epimer
(that is peak 4 = 20S-epimer of the
hexanor-1,20-(OH)D, peak 6 =
24R-hydroxy OCT, and peak 7 = 25R-form of
26-hydroxy OCT). All chemically synthesized standard compounds were
subjected to GC-MS for comparison to previously obtained mass spectra
of biological products. Mass spectra (data not shown) were found to be
virtually identical to those shown in Fig. 2Fig. 3Fig. 4Fig. 5and therefore
corroborate the identifications provided.
Time Course of OCT Metabolism in HPK1A-ras Cells
Evidence from the time course suggests that at early time
points the hexanor-1,20-(OH)D is present when
there is no detectable hexanor-20-oxo-1-OH-D (Fig. 6). All other metabolites accumulate at early time
points, and their concentration plateaus or even decreases by the end
of the incubation period (24 h).
Studies of the Metabolism of OCT in the HPK1A
Keratinocyte, the Rat Osteosarcoma, UMR-106, and the Human Hepatoma,
HepG2
In these studies we examined the metabolism of OCT using
established cell lines involved in both the activation and catabolism
of vitamin D. With such cell lines it was found that OCT was
metabolized to a smaller number of products, which comigrated with
peaks 4, 6, and 7 obtained using HPK1A-ras (Fig. 7).
Metabolites were subjected to the same rigorous identification used for
those from HPK1A-ras.
Biological Activities of OCT and Its Metabolites
The VDR binding affinities of the 26-hydroxy-OCT isomers
(ED141 and ED142) or side chain truncated metabolites of OCT (ED26,
ED190, and ED67) were much reduced compared with the parent molecule,
OCT, which showed approximately the same binding affinity for the
mammalian VDR as 1,25-(OH)D (Fig. 10A). This was especially true of the side
chain truncated metabolites of OCT, which showed a binding affinity
2-3 orders of magnitude lower than
1,25-(OH)D. On the other hand,
24R-hydroxy-OCT (ED106) or 24-oxo-OCT (ED174) retained much of
the VDR-binding affinity of OCT with values only - that of OCT,
respectively.
, 25-(OH)D). A,
VDR binding. B, DBP binding. C, inhibition of HL-60
cell growth. For clarity, only the chemically synthesized epimer
corresponding to the biologically generated metabolite is illustrated.
The biological activity of the other epimer was determined, and the
data are depicted numerically in Fig. 11. 
,
1,25-(OH)D; 
, OCT; 
,
24R-OH-OCT; 
, (25R)-26-OH-OCT; 
,
hexanor-20-oxo-1-OH-D; 
,
hexanor-1,20S-(OH)D.
,25-(OH)D and 4 orders of
magnitude lower than 25-OH-D (data not shown). No
metabolite of OCT possessed a higher affinity for DBP than OCT.,25-(OH)D suppressed cell growth of HL-60
in a dose-dependent manner when used at a concentration between 1
10M and 1 10M. Significant inhibition of growth could be seen at a
steroid concentration as low as 10
M 1,25-(OH)D where the growth was
decreased to <40% of control. The suppression of cell growth
provided by OCT was virtually identical, whereas the suppressions
caused by the hexanor-20-oxo-compound (ED26) or the
hexanor-20S-hydroxy- compound (ED67) were greater than 2 orders of
magnitude less effective than 1,25-(OH)D.
Other metabolites such as 24R-hydroxy-OCT (ED106) or
(25R)-26-hydroxy-OCT (ED141) showed slightly inferior
growth-inhibiting activity to OCT, with potencies between 
and
that of 1,25-(OH)D. The results of the
biological activity studies for all chemically synthesized epimers,
including biologically generated products of OCT, are summarized in Fig. 11.
,25-(OH)D.
Our study is the first to rigorously identify any of the metabolic
products of OCT formed in vitro, and this was made possible by
the generation of microgram quantities of these metabolites. The novel
metabolites identified include two truncated versions of the OCT
molecule lacking carbons 23-27 and containing a 20-oxo or
20-hydroxyl group. The 20-hydroxy compound has been reported before in vitro, but identification was based solely upon comigration
with a chemically synthesized standard on HPLC(11) . Here we
were able to augment the preliminary identification afforded by HPLC
co-chromatography with GC-MS analysis and later comparison with
chemically synthesized standards. Furthermore, our work is also
consistent with the preliminary finding of a glucuronide conjugate of
the 20-hydroxy compound in rat bile following OCT
administration(10) . The 24R-OH-OCT and 26-OH-OCT in
the 25R-form identified here are novel, and their
stereochemistry is also established.-OH-D in the
same hepatoma cell line HepG2 (23) . In that case, we also
found cleaved molecules, although the ultimate products were
23-substituted and lacked the carbon atoms C-25, C-26, and C-27 of the
starting material. Thus, in the cases of both 22-oxacalcitriol and
24-oxa-1-hydroxyvitamin D, the molecule is cleaved
just distal to the oxygen atom. In the case of
24-oxa-1-OH-D, we hypothesized that the enzymatic
hydroxylation of a carbon vicinal to the oxygen atom would give rise to
an unstable hemiacetal linkage that would undergo spontaneous breakdown
to the C-23 substituted products(23) . If we extrapolate from
this result to the case of OCT we would predict that C-23 hydroxylation
would have to occur in order to generate an unstable hemiacetal and
truncated products retaining the 22-oxa group. The metabolites that
were observed, namely 24-OH-OCT and 26-OH-OCT, would appear to be
either precursors to this 23-hydroxylated intermediate or are
by-products of the metabolic machinery. It is interesting to note that
a 24-hydroxylated version of 1,25-(OH)D precedes a 23-hydroxylated metabolite in the catabolic sequence
for the natural hormone during its conversion to calcitroic acid
observed in a variety of target cell systems(19, 24) .
Alternatively, others have identified 26-hydroxylated derivatives of
25-OH-D(25) and 1,25-(OH)D(26) and a 26-hydroxylated metabolite is observed as an
intermediate during formation of the 26,23-lactone, but this is
postulated to occur after and not prior to
23-hydroxylation(27) . It remains to be proven if either
24-OH-OCT or 26-OH-OCT is a precursor to the 20-hydroxy and 20-oxo
derivatives also found here.
-H]OCT (12 Ci/mmol) was incubated at low
nanomolar concentrations with HPK1A-ras cells (data not
shown). The formation of the 20-oxo derivative could also be due to the
different redox state of these cells compared with the other cell lines
used. On the other hand, this may provide evidence of the role of one
compound as an intermediate in the formation of the other (see Fig. 9). The time course study carried out suggests that the
20-hydroxy compound predates the 20-oxo derivative and thus may be
converted to the latter in this culture system.,25-(OH)D) in all parameters measured (i.e. VDR binding, DBP binding, and growth suppression of
HL-60). This was particularly marked for the 20-oxo and 20-hydroxy
derivatives where truncation of the side chain of OCT lowered
biological activity by more than 2 orders of magnitude. A rough
correlation between the strength of calf thymus VDR binding and the
ability of the vitamin D analog to inhibit growth of HL-60 cells was
still discernible. As with other 22-oxa analogs studied in this field (e.g. 28), the calcium-transporting activity of the
metabolites reported here remains unknown.,25-(OH)D metabolism(19) , OCT metabolism is vitamin D-inducible in
cultured bovine parathyroid cells (11) and normal human
peripheral monocytes (29) . The result that hepatoma cells are
also able to form truncated OCT metabolites is interesting given that
liver cells are thought to be devoid of the vitamin D receptor and the
vitamin D-inducible catabolic enzymes (e.g. cytochrome P-450
CYP24)(30, 31) . However, small modifications of the
vitamin D side chain have been shown to result in much more rapid
hepatic metabolism and indeed 24-hydroxylation and 24-oxidation of
certain analogs (e.g. calcipotriol)(32, 12, 15) . Thus, it is
possible that in addition to being susceptible to attack by vitamin
D-inducible enzymes in classical vitamin D target cells, OCT is also
attacked by more general enzymes in the liver leading to cleavage of
the side chain by similar or slightly different mechanisms. It is clear
from the studies presented here that OCT is more efficiently degraded
by vitamin D-target cells than by hepatoma cells, and this may be
important to the in vivo distribution and biological activity
of the drug (33) as well as to the endogenous
ligand(34) .
) and D indicate vitamins D and D, respectively,
and OH and (OH) indicate hydroxy and dihydroxy,
respectively. Therefore, for example, 1,25-(OH)D represents 1,25-dihydroxyvitamin D.)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
