From the
A series of homologated 1
In recent years there has been an explosion in the number of
novel analogs of vitamin D
Homologated vitamin D metabolites
with a single extra carbon (C-24a) between carbons C-24 and C-25 or on
the C-26 and/or C-27 were first synthesized by Ikekawa and co-workers
and tested biologically by DeLuca and co-workers (8, 9, 10) and by Stern's
group(11) . It was quickly recognized that these homologated
analogs might offer potential as drugs because of increased biological
activity in several in vitro assays. However, the exact
molecular basis for this increased biological activity was never fully
elucidated, and the metabolic fate of these homologated analogs never
studied. The availability of a series of such homologated vitamin D
analogs together with much biological data made them particularly
attractive for metabolic study using a systematic approach.
Furthermore, the recent clinical research interest in the use of more
complex homologated vitamin Ds (e.g. EB1089 and KH1060, Leo
Pharmaceutical Products) made the choice of metabolism of homologated
vitamin D analogs a topical subject for investigation.
The vitamin D
hydroxylases constitute a family of mixed-function oxidases which
contain as their specific component, a cytochrome P450 isoform. Two
such cytochrome P450 isoforms have been cloned, one putatively
representing the liver vitamin D
Thus, in these studies, we set out to use
the homologated series of analogs as tools in the study of the two best
known hydroxylases, the liver vitamin D
Further steps of HPLC were performed on the crude peaks isolated
from the first step of purification, using a Zorbax-CN (0.46
Co-chromatography of
each purified biologically derived metabolite with its chemically
synthesized counterpart was attempted using one of the two HPLC column
systems described above. The inclusion of 1
The major metabolites were identified
by GC-MS and direct comparison with chemically synthesized standards
(see ). All three putative 23-hydroxylated metabolites
exhibited identical relative retention times on HPLC to one of the two
23(R)- or 23(S)- chemically synthesized epimeric
forms. The metabolite, MC1127-MetC (RRT = 3.52 ± 0.02)
co-migrated with 23(R)-OH-MC1127 (RRT = 3.56 ±
0.01) but not 23(S)-OH-MC1127 (RRT = 3.44 ±
0.01); the metabolite MC1147-MetC (RRT = 3.50 ± 0.02)
co-migrated with 23(S)-OH-MC1147 (RRT = 3.59 ±
0.04) but not 23(R)-OH-MC1147 (RRT = 3.75 ±
0.03); the metabolite MC1179-MetC (RRT = 4.41 ± 0.02)
co-migrated with 23(R)-OH-MC1179 (RRT = 4.43 ±
0.03) but not 23(S)-OH-MC1179 (RRT = 4.23 ±
0.06) (see for column and solvent systems used). Since the
synthetic reference 24-hydroxylated standards ran as single peaks on
HPLC and GC, identification of R/S isomers was not possible.
However, relative retention times on HPLC of MC1127-MetA, MC1147-MetD,
and MC1179-MetB were similar to those of the diastereoisomeric mixtures
of their respective 24-hydroxylated standards. These were as follows:
MC1127-MetA (RRT = 2.97 ± 0.01 min) compared with
24-OH-MC1127 (RRT = 2.99 ± 0.01); MC1147-MetD (RRT
= 3.22 ± 0.02) compared with 24-OH-MC1147 (RRT =
3.21 ± 0.06); MC1179-MetB (RRT = 3.59 ± 0.04)
compared with 24-OH-MC1179 (RRT = 3.65 ± 0.02) (see for column and solvent systems used).
The identity of
the 23-hydroxylated metabolites MC1127-MetC, MC1147-MetC, and
MC1179-MetC were confirmed by comparing the mass spectra of
pertrimethylsilylated ether derivatives of the putative metabolites
with those of chemically synthesized standards (Fig. 3). In each
case, the mass spectrum obtained from the biologically generated
metabolite is very similar to that of its chemically synthesized
standard (essentially no differences between R/S versions were
observed). Molecular ions of m/z 734, 748, and 762 were
observed for the pertrimethylsilylated ether derivatives of
MC1127-MetC, MC1147-MetC, and MC1179-MetC, respectively, as expected
for hydroxylation of the starting compounds. Ions of m/z 171,
185, and 199 result from the loss of a single silanol group from side
chain fragments m/z 261, 275, and 289 for MC1127-MetC,
MC1147-MetC, and MC1179-MetC, respectively, which arise from cleavage
of the C-22-C-23 bond and suggest C-22-C-23 fragility. The
mass spectra of all three 23-hydroxylated metabolites contained ions at m/z 395 and 305 due to the sequential loss of two and three
silanol groups from the predicted ion m/z 575 (M - 159,
M - 173, or M - 187, respectively) common to all
23-hydroxylated metabolites. This fragmentation pattern again suggests
C-23-C-24 fragility. As with MC1147-MetB, an ion of m/z 278 was observed in all three spectra. This ion probably arises
from a cleavage of the bond between C-22 and C-23 resulting in the loss
of 261, 275, or 289 mass units from the molecular ion of each
23-hydroxylated metabolite, respectively. The resultant fragment of m/z 473 loses a methyl group and 2 silanols to give rise to
the fragment at m/z 278. These results further confirm the
identity of MC1127-MetC, MC1147-MetC, and MC1179-MetC as
23(R)-OH-MC1127, 23(S)-OH-MC1147, and
23(R)-OH-MC1179, respectively.
In this paper we have used two in vitro cultured
cell models to compare and contrast the substrate preferences of the
cytochromes P450 responsible for 25-hydroxylation of vitamin D
Although the cytochromes P450 involved in these liver and target
cell systems are consistent in their site of hydroxylation and are
tolerant of small changes in the vitamin D side chain, we did observe
some modification in their efficiency. The ratio of 25:27 hydroxylation
changes as the side chain is extended. Since these two hydroxylations
are carried out by the same cytochrome, CYP27, and since this ratio is
altered when other changes are made to the substrate (e.g. using a bile acid precursor with an intact steroidal ring
structure or using a vitamin D
These findings have implications for modelling of the substrate
binding pocket of the vitamin D-related cytochrome P450 isoforms and
for the physiological role of these enzymes. The liver mitochondrial
cytochrome P450, CYP27, seems to be a broad specificity enzyme
tolerating not only extension of the side chain as we have observed
here, but also greatly modified steroidal/vitamin D nucleus (15, 16, 30) and 20-epimerization of the side
chain(31) . Taken together, these data suggest that the
substrate binding pocket of CYP27 can tolerate a variety of steroidal
shapes either by accommodating only the terminal carbons of the side
chain or by having a specific cleft for the side chain within a broader
pocket for the vitamin D/steroidal ring structure (Fig. 9).
Whereas in
this discussion we have concentrated on the investigation of the
regioselectivity of the hydroxylating enzymes, the question of their
stereoselectivity remains to be addressed. The availability of both
23R- and 23S-hydroxylated standards has enabled us to
make the inference that only one of the two isomers is formed in
substantial amounts in each case. However, the alternating pattern that
begins to emerge (23R-hydroxylation of MC1127,
23S-hydroxylation of MC1147, 23R-hydroxylation of
MC1179) is a puzzling result but is reminiscent of the 23S,
24R, 25S pattern pointed out by Ikekawa(7) .
Identification of the 24-configuration of the 24-OH metabolites and the
25-configuration of the 27-OH metabolites awaits further work.
This
study has firmly established the value of performing metabolic studies
of vitamin D analogs in vitro using cultured cells. The
convenient incubation of cells with vitamin D analogs permits the
isolation of nanogram to microgram quantities of metabolic products
which can be rigorously identified with the aid of GC-MS techniques and
comparison to chemically synthesized standards.
We acknowledge the important contribution of Dr. Johng
Rhim, NCI, National Institutes of Health, Bethesda, MD, in the
development of the HPK1A-ras cell line used in some aspects of
this work.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-hydroxyvitamin D
and
1,25-dihydroxyvitamin D
molecules with one to three extra
carbons in the side chain were used to examine the substrate
preferences and hydroxylation site selection mechanisms of the liver
vitamin D
-25-hydroxylase (CYP27) and the target cell
25-hydroxyvitamin D
-24-hydroxylase (CYP24). Cultured and
transfected cell models, used as sources of these hydroxylases, gave
23-, 24-, 25-, and 27-hydroxylated metabolites which were identified by
their high performance liquid chromatography and GC-MS characteristics.
Lengthening the side chain is tolerated by each cytochrome P450 isoform
such that 25-hydroxylation or 24-hydroxylation continues to occur at
the same rate as in the native side chain, while the site of
hydroxylation remains the same for the liver enzyme in that CYP27
continues to hydroxylate at C-25 and C-27 (minor) despite the
two-carbon-atom extension. Somewhat surprising is the finding that C-24
and C-23 (minor) hydroxylations also do not change as the side chain is
extended by as much as three carbons. We conclude that CYP24 must be
directed to its hydroxylation site(s) by the distance of carbon 24 from
the vitamin D ring structure and not as in CYP27 by the distance of the
hydroxylation site from the end of the side chain.
chemically synthesized for the
purpose of developing drugs for the treatment of metabolic bone
disease, cancer, and psoriasis(1) . In large part, this recent
interest in vitamin D analogs arose from the finding that the hormonal
form, 1,25-dihydroxyvitamin D
(1,25-(OH)
D
),
(
)stimulates differentiation of certain cells in the skin and
myeloid lineages as well as performing its classical roles in
Ca
and PO
homeostasis(2) . Despite the fact that many of these
analogs have been screened for biological activity in a variety of
assays (calcemic activity, differentiation activity, vitamin D receptor
binding, vitamin D binding globulin binding), few have been subjected
to pharmacokinetic analysis (3). Even fewer have been subjected to
detailed metabolic studies. Exceptions include MC903 (calcipotriol) (4, 5) and 22-oxa-calcitriol(6) ,
(
)but in these studies the principal focus has been
defining the nature of the metabolic products for governmental drug
approval. We know of no example where a series of analogs have been
used to define the substrate requirements of the hydroxylase enzymes
involved in vitamin D metabolism.
-25-hydroxylase (CYP27) and
the other that of the kidney 25-OH-D
-24-hydroxylase
(CYP24)(12, 13) . Few experiments have been carried out
using the expression of the cDNAs of these cytochrome P450s to
determine their specificity, but other studies have shown that CYP27
has broad substrate specificity, hydroxylating bile acid intermediates
and a range of vitamin D compounds(14, 15) .
Furthermore, our own laboratory has shown CYP27 to lack
regiospecificity, placing hydroxyl groups into a number of carbon
positions at C-24, C-25, and C-27 of the classical D
and
D
side chains(16) . No studies have been performed
with the newer analogs to probe the structural requirements of these
cytochrome P450 isoforms.
-25-hydroxylase and
the target cell 25-OH-D
-24-hydroxylase. We chose to study
metabolism in vitro using cultured cell systems we have
established for this purpose (17, 5) and, in the case of
CYP27, confirmed cell culture findings with cDNA transfection
studies(16) . Our findings show that the homologated vitamin D
analogs are extremely valuable in that they reveal important
information about how the two enzymes interact with their substrates.
Materials
The human keratinocyte cell line,
HPK1A (18) transformed with the ras oncogene,
HPK1A-ras(19) was developed previously in
collaboration with Dr. Johng Rhim (National Cancer Institute).
Similarly, the development of the human liver cell line, HH01, by one
of us (E. R.) has been described previously(20) . The SV-40
transformed African Green Monkey kidney cell line, COS-1, was purchased
from the American Tissue Culture Collection. All solvents used were of
HPLC grade and were obtained from Caledon Laboratories (Georgetown, ON,
Canada). The bovine adrenodoxin expression plasmid pBAdx-4 (21) was a gift of Dr. M. Waterman (University of Texas Medical
School, Dallas, TX).
Synthesis of Vitamin D Analogs
The vitamin D
analogs: 24a,24b-dihomo-1-(OH)D
(MC1281),
24a-homo-1
,25-(OH)
D
(MC1127),
24a,24b-dihomo-1
,25-(OH)
D
(MC1147), and
24a,24b,24c-trihomo-1
,25-(OH)
D
(MC1179) (Fig. 1) were synthesized as described previously(22) .
Their 23- and 24-hydroxylated derivatives were synthesized by methods
to be described elsewhere. The 23-hydroxylated compounds were available
separately with known configuration, whereas the 24-hydroxylated
compounds were available as 1:1 diastereoisomeric mixtures. The
radioactive hormone,
[1
-
H]1,25-dihydroxyvitamin D
([
H]1,25-(OH)D
) was synthesized
as described previously(23) .
Figure 1:
Structures of
homologated vitamin D analogs. Structures of the side chains of
1,25-(OH)D
analogs and 1
-OH-D
analogs, together with the ring structure of both types of
vitamin D analogs.
Generation of Metabolites of MC1127, MC1147, and MC1179
Using HPK1A-ras Cells
Metabolites were generated from
HPK1A-ras cells as described
previously(5, 24) . Cells were maintained in 150-mm
plates using Dulbecco's culture medium (Dulbecco's modified
Eagle's medium). Near confluence, cells were treated with
1,25-(OH)D
(10 nM) to induce
transcription of catabolic enzymes. After 18 h, the medium was replaced
by Dulbecco's modified Eagle's medium supplemented with 1%
BSA and 100 µM DPPD (Sigma). Cells were then incubated for
48 h in the presence of vehicle (0.01% EtOH) or a 10 µM concentration of analog in vehicle.
Generation of Metabolites of MC1281 Using HH01
Cells
HH01 were maintained in -MEM supplemented as
described previously(20) . Near confluence, the medium was
replaced with
-MEM supplemented with 1% BSA and 100 µM DPPD. Cells were incubated in the presence of vehicle (0.01% EtOH)
or 25 µM MC1281 in vehicle for 20 h.
Incubations of MC1281 with CYP27-transfected COS-1
Cells
COS-1 were transfected with the expression vectors
pSG5-CYP27 and pBAdx (adrenodoxin) using the standard DEAE-Dextran
method (25) as described previously(16) . Forty-eight
hours post-transfection, cells were washed twice with
phosphate-buffered saline, and the medium was replaced with
Dulbecco's MEM supplemented with 1% BSA and 100 µM DPPD. Cells were then incubated for 30 h in the presence of
vehicle (0.01% EtOH) or MC1281 (25 µM) in vehicle.
Purification of Metabolites
Cells and medium were
extracted as described previously(24) . HPLC separation of
metabolites was achieved using a Zorbax-SIL (0.62 8 cm; 3
µ) column eluted with HIM 91:7:2 at a flow rate of 1 ml/min.
Metabolites were identified based on their characteristic vitamin
D
chromophore (UV
= 265, UV
= 228, UV
/UV
= 1.75).
25
cm; 6 µ) column and 91:7:2 (HIM) solvent system at a flow rate of 1
ml/min. Two rounds of HPLC yielded metabolites which were pure on the
basis of the homogeneity of the collected peak.
-OH-D
as an
internal standard allowed us to convert retention times of metabolites
into relative retention times, and mean relative retention times were
calculated from triplicate HPLC runs. Mean relative retention times
were considered identical if they were within two standard deviations
of each other.
Chemical Modification of Metabolites
Purified
metabolites were subject to chemical modification using sodium
metaperiodate or sodium borohydride as described
previously(24) . Modified metabolites were subject to HPLC
analysis using the system described above.
GC-MS
Purified metabolites were derivatized to
pertrimethylsilyl ethers, then analyzed by gas chromatography-mass
spectrometry (GC-MS) as described previously(26) . Injection of
metabolites of analogs described in this paper into the high
temperature injection zone of the GC causes B ring closure producing
pyro- and isopyro- isomers. Mass spectra were obtained only from the
pyro- isomer, 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 pyro- isomer. Mass
spectra were obtained by averaging each peak and subtracting the
background.
Catabolism Rate Assay
The rate of metabolism of
the 1,25-(OH)D
analogs was measured as
described previously(24) . HPK1A-ras cells were
incubated with [
H]1,25-(OH)
D
(23 nM) in the presence or absence of varying
concentrations of analog (0 to 23 µM) for 3 h at 37
°C. Triplicate 500-µl aliquots of aqueous fraction from the
cell/medium extract were mixed with aqueous scintillation mixture, and
the radioactivity was measured using a scintillation counter.
25-Hydroxylation Rate Assay
The rate of metabolism
of 1-(OH)D
and MC1281 were compared using a time
course study. The cell line HH01 was grown in 6-well plates as
described above. Near confluence, the medium was replaced with
-MEM supplemented with 1% BSA and 100 µM DPPD. Cells
were incubated with 25 µM analog for 0, 6, 12, or 18 h,
then extracted, and purified as described above. Both the amount of
substrate remaining and metabolites produced were determined by
integrating the area under peak of UV absorbance at 265 nm. Each data
point is the average of three incubations.
Generation of Metabolites from
1,25-(OH)
Incubation of 1,25-(OH)D
in HPK1A-ras
Cells
D
with
HPK1A-ras cells resulted in the formation of the four
lipid-soluble metabolites of the hormone (Fig. 2D). The
four metabolites generated include 1,24,25(OH)
D
(peak D), 24-oxo-1,25-(OH)
D
(peak A),
24-oxo-1,23,25(OH)
D
(peak C), and
24,25,26,27-tetranor-1,23(OH)
D
(peak B) as
determined by co-migration with standard compounds (data not shown).
These metabolites correspond to the intermediates which have previously
been reported to be a part of the C-24 oxidation pathway of vitamin
D
(27) . This confirms that the Ha-ras transformed cell line, HPK1A-ras, expresses functional
enzymes involved in the side chain oxidation pathway for inactivation
of 1,25-(OH)
D
.
Figure 2:
HPLC profiles of lipid extracts from
HPK1A-ras cells incubated with homologated vitamin D analogs.
Total lipid extracts were separated on a Zorbax-SIL column using the
solvent HIM 91:7:2 at a flow rate of 1 ml/min. Metabolites of MC1127 (A), MC1147 (B), MC1179 (C), and
1,25-(OH)D
(D) were identified based
on their characteristic vitamin D chromophore (UV
= 265 nm, UV
= 228 nm,
UV
/UV
= 1.75) and have been shaded. Only the region of the chromatogram containing vitamin
D metabolites is depicted. The inset in B shows the
subsequent separation of metabolites MC1147-MetC and MC1147-MetD during
Zorbax-CN rechromatography of the 19.8-22-min peak isolated from B.
Generation of Metabolites from Homologated Analogs in
HPK1A-ras Cells
Analytical HPLC separation of lipid soluble
HPK1A-ras cell extracts taken from cells incubated with
homologated analogs (10 µM) indicated that we were able to
generate three (or more) metabolites from MC1127 (Fig. 2A) and MC1147 (Fig. 2B), while
only two metabolites were generated from MC1179 (Fig. 2C). Rechromatography of the peak labeled
MC1147-MetC (see inset in Fig. 2B) indicated
that this peak was a mixture of two metabolites which could be resolved
on Zorbax-CN (termed peaks C and D). As we were able to generate only
two metabolites from MC1179, a second incubation was performed in which
the ratio of cultured cell density:concentration of substrate (MC1179)
was increased in order to generate a greater amount of less abundant
metabolites. In doing so, we were able to generate sufficient
quantities of this third metabolite, termed MC1179-MetC (peak not
apparent in the chromatogram shown in Fig. 2C), to be
identified later by GC-MS. The chromatographic properties of the
purified metabolites generated from all three analogs are summarized in . The availability of reference standards of both R and S forms allowed the exclusion of the presence of
other isomers in the extracts.
Figure 3:
Mass spectra of putative 23-hydroxylated
metabolites. TMSi derivatives of the purified metabolites MC1127-MetC (top left), MC1147-MetC (top middle), and MC1179-MetC (top right) and the synthetic standards
23(R)-OH-MC1127 (bottom left),
23(S)-OH-MC1147 (bottom middle), and
23(R)-OH-MC1179 (bottom right) were analyzed by GC-MS
under conditions described in the text. In each case, spectra represent
the pyro- isomer derived from each metabolite and for convenience
spectral interpretation is given with reference to the parent
metabolite.
To confirm the identities of
MC1127-MetA, MC1147-MetD, and MC1179-MetB as the 24-hydroxylated
metabolites, the mass spectra of pertrimethylsilylated ether
derivatives of the purified metabolites were again compared to those of
the chemically synthesized standards (Fig. 4). The mass spectra
of the pertrimethylsilylated ether derivatives of the putative
24-hydroxylated metabolites are very similar to those of their
corresponding chemically synthesized standards. In each case, molecular
ions at m/z 734, 748, and 762 for the derivatives of
MC1127-MetA, MC1147-MetD, and MC1179-MetB are consistent with the
hydroxylation of the substrate. Ions of m/z 157, 171, and 185
due to the loss of a single silanol from side chain fragments of m/z 247, 261, and 275 are observed for MC1127-MetA,
MC1147-MetD, and MC1179-MetB, respectively, and indicate
C-23-C-24 bond fragility. The mass spectra of all three
24-hydroxylated metabolites contained ions of m/z 499 and 409
due to the sequential loss of one and two silanol groups, respectively,
from the predicted m/z 589 (M - 145, M - 159, or M
- 173) fragment common to all 24-hydroxylated metabolites. This
fragmentation pattern suggests C-24-C-24a bond fragility. Also
observed in the mass spectra of all three 24-hydroxylated metabolites
was an ion at m/z 292, which is probably generated by cleavage
of the bond between C-23 and C-24 resulting in the loss of 247, 261, or
275 mass units from the molecular ion of each 24-hydroxylated
metabolite, respectively. The resultant fragment of m/z 487
loses a methyl group and 2 silanols to give the fragment at m/z 292. We conclude on the basis of these results that MC1127-MetA,
MC1147-MetD, and MC1179-MetB are the 24-hydroxylated metabolites of
their respective analogs.
Figure 4:
Mass spectra of putative 24-hydroxylated
metabolites. TMSi derivatives of the purified metabolites MC1127-MetA (top left), MC1147-MetD (top middle), and MC1179-MetB (top right) and the synthetic standards 24-OH-MC1127 (bottom left), 24-OH-MC1147 (bottom middle), and
24-OH-MC1179 (bottom right) were analyzed by GC-MS under
conditions described in the text. In each case, spectra represent the
pyro- isomer derived from each metabolite and for convenience spectral
interpretation is given with reference to the parent
molecule.
The minor metabolites illustrated in Fig. 2and listed in , MC1127-MetB, MC1147-MetB, and
MC1179-MetA were all found to be sodium borohydride-sensitive, and
their mass spectral and chromatographic properties (see )
are consistent with the presence of oxo-groups at C-23 or C-24. One
other metabolite, MC1147-MetA, was studied but not conclusively
identified on the basis of the information gathered (see ).
Rate of 24-Hydroxylation of Homologated
Analogs
The effect of lengthening the vitamin D side
chain on the rate of 24-hydroxylation was also examined using
HPK1A-ras cells (Fig. 5). Measuring the ability of the
analogs to compete with
[1
-
H]1,25-(OH)
D
for
the enzymes of the side chain oxidation pathway, we observe that
extending the vitamin D
side chain does not significantly
alter the rate of 24-hydroxylation.
Figure 5:
Competitive inhibition of
[1-
H]calcitroic acid production using
nonradioactive homologated 1,25-(OH)
D
analogs
in HPK1A-ras cells.
[1
-
H]1,25-(OH)
D
was
incubated in HPK1A-ras cells in the presence of vehicle alone
or varying concentrations of MC1127 (▾), MC1147 (
), MC1179
(
), or 1,25-(OH)
D
(
). Incubations
were performed as outlined under ``Experimental Procedures.''
Each point in the figure is the mean ± S.E. of three flasks
counted in triplicate.
Generation of Metabolites from Homologated
1
Northern analysis of
HH01 cells using a CYP27 cDNA probe (16) indicated that CYP27
mRNA was expressed in this cell line (data not shown). Incubation of
HH01 cells with either 1-OH-D
Analog
-OH-D
or MC1281 in each case
resulted in the generation of two metabolites (Fig. 6, A and B). Other studies have shown that the vitamin D
metabolites formed by HH01 cells are generated by cytochrome
P450-dependent steps as judged by their sensitivity to heat
inactivation and their total inhibition by the anti-fungal agent
ketoconazole (used at a concentration of 5 µM).
(
)The two products of 1
-OH-D
(MetA and
MetB) have been previously identified as 1,25-(OH)
D
and 1,26(27)-(OH)
D
,
respectively(16) . In the case of MC1281, the less polar of the
two metabolites (MC1281-MetA) co-migrated with the synthetic standard
24a,24b-dihomo-1,25-(OH)
D
(MC1147) on two
separate HPLC systems ().
Figure 6:
HPLC profiles of lipid extracts from HH01
cells incubated with 1-OH-D
or MC1281. Total lipid
extracts were separated on a Zorbax-SIL column using the solvent HIM
91:7:2 at a flow rate of 1 ml/min. Metabolites of 1
-OH-D
(A) or MC1281 (B) were identified based on
their characteristic vitamin D chromophore (UV
=
265 nm, UV
= 228 nm, UV
/UV
= 1.75) and have been shaded.
The metabolites generated in
HH01 cells were further characterized by GC-MS analysis. The mass
spectra of the pertrimethylsilylated ether derivatives of MC1281-MetA
and the chemically synthesized standard
24a,24b-dihomo-1,25-(OH)D
(MC1147) are shown in Fig. 7. Both spectra show a molecular ion at m/z 660 and
a major fragment at m/z 131 corresponding to
[(CH
)
C=OTMS]
produced by cleavage of the bond between C-24 and C-25. Such a
fragment at m/z 131 is characteristic of most 25-hydroxylated
vitamin D derivatives (e.g. 25-OH-D
). This
evidence confirms the identify of MC1281-MetA as the 25-hydroxylated
derivative. The mass spectrum of the pertrimethylsilylated ether
derivative of MC1281-MetB also showed a molecular ion of m/z 660, again suggesting hydroxylation of MC1281. However, the
appearance of a fragment at m/z 103 and none at m/z 131 indicates that hydroxylation has occurred at a different site,
probably C-27 in this case, the fragment at m/z 103
representing [C(27)-OTMS]
and being derived
from C-25-C-27 cleavage. Thus the data are consistent with the
minor metabolite, MC1281-MetB being the 27-OH derivative.
Figure 7:
Mass spectra of the putative
25-hydroxylated metabolite MC1281-MetA formed in HH01 cells and its
chemically synthesized standard. TMSi derivatives of the purified HH01
metabolite MC1281-MetA from Fig. 6B was compared to synthetic
25-OH-MC1281 (also known as MC1147) on GC-MS as described in the text.
In each case, spectra represent the pyro- isomer derived from each
metabolite, and, for convenience, spectral interpretation is given with
reference to the parent molecule.
The rate
of metabolism of the side-chain-lengthened analog was compared to that
of 1-OH-D
in HH01 cells. Measuring either the
generation of metabolites (Fig. 8) or the disappearance of
substrate (Fig. 8, inset), we observe that the rate of
metabolism of MC1281 is not significantly different from that of
1
-OH-D
.
Figure 8:
Time course of the metabolism of
1-OH-D
and MC1281 in HH01 cells. Cells were incubated
with 15 µg of analog for various times between 0 and 18 h. The
graph depicts the amount of product (25- and 27-hydroxylated) formed;
the inset shows the amount of substrate metabolized versus time. Metabolites were quantitated based on integration of UV
peaks at an absorbance of 265 nm. Each point is the average ±
S.E. of three incubations.
To confirm that the two metabolites
generated from MC1281 in HH01 cells were oxidation products mediated by
the cytochrome P450 CYP27, MC1281 was incubated with COS-1 cells
transfected with the cDNAs encoding CYP27 and adrenodoxin. Incubation
of MC1281 with the transfected COS-1 cells resulted in the generation
of two metabolite peaks that were absent in control transfected COS-1
cells. On HPLC, the two putative CYP27-generated products possessed
similar retention times to metabolites MC1281-MetA and MC1281-MetB
generated in HH01 cells (data not shown).
in the liver and the 23- and 24-hydroxylation of
1,25-(OH)
D
in vitamin D target cells.
Specifically, we have shown that lengthening the side chain of the
vitamin D molecule is tolerated quite well in that each enzyme is able
to continue to hydroxylate efficiently. However, most interestingly,
our results reveal that the two cytochromes P450 must recognize their
respective sites of hydroxylation by different mechanisms: the
25-hydroxylase appears to be directed to its terminal hydroxylation
site by the distance from the end of side chain, whereas the 23- and
24-hydroxylases appear to be positioned at their hydroxylation site by
the distance from the vitamin D nucleus. As a result and in accord with
the recent studies of the liver side-chain hydroxylation of a variety
of vitamin D compounds with the conventional D
and D
side chains(16) , 25- and 27-hydroxylation of
24a,24b-dihomo-1
-OH-D
can be demonstrated to occur in
the HH01 cell line. However, using the target cell side-chain
hydroxylation system (HPKIA-ras), we continued to observe 23-
and 24-hydroxylation of 24a-homo, 24a,24b-dihomo, and
24a,24b,24c-trihomo-1,25-(OH)
D
derivatives even
with the addition of 1-, 2-, and 3-carbon extensions to the side chain.
In other words, the 23- and 24-hydroxylases did not move up the side
chain to C-24 and C-24a of MC1127 or C-24a and C-24b of MC1147; or
C-24b and C-24c of MC1179 as might have been expected if the 23- and
24-hydroxylase enzyme(s) had recognized its (their) site of
hydroxylation based upon the distance from the end of the side chain.
The experimental basis for these conclusions are well supported by
careful and rigorous identification of side-chain-hydroxylated
metabolites by HPLC, GC-MS, and chemical derivatization techniques. The
identifications are further reinforced by comparisons to a series of
23- and 24-hydroxylated standards chemically synthesized for this
purpose. It is clear that the 23- and 24-positions remain the primary
hydroxylation sites for all members of the homologated series.
side chain), this finding is
not surprising. Transfection studies reported here lend support to the
concept that CYP27 is responsible for most 25- and 27-hydroxylations of
steroidal substrates in HH01 liver cells. CYP27 gives a pattern of
products similar to that seen in HH01 cells. The efficiency of
23-/24-hydroxylation of homologated 1,25-(OH)
D
analogs is similarly well maintained despite a greater degree of
chain extension. However, at the extreme of C
addition to
the side chain in 24a-, 24b-, and
24c-trihomo-1,25-(OH)
D
, we observe reduced
23-hydroxylation of the side chain and a much increased ratio of
24:23-hydroxylation suggesting that this side chain is less well
accommodated by the active site of the enzyme or enzymes involved. It
is now well established that 24-hydroxylation is carried out in vitamin
D target cells by CYP24, a cytochrome P450 distinct from the other
families(13) . Not so obvious is whether 23-hydroxylation can be
carried out by a separate cytochrome P450 or is another product of
CYP24. Early studies of CYP24 did not reveal catalytic properties in
addition to 24-hydroxylation(28) . However, more recently,
Akiyoshi-Shibata et al.(29) showed that a bacterially
expressed CYP24 was active in a reconstitution system and was capable
of the generation of 24-oxo metabolites and 24-oxo-23-hydroxylated
metabolites as well as 24-hydroxylated metabolites from vitamin D
substrates. These same products of CYP24 were previously proposed to
lie on a C-24 oxidation pathway purported to function in the catabolism
of 1,25-(OH)
D
to calcitroic acid(23) .
In their work on CYP24, Akiyoshi-Shibata et al.(29) did not observe the direct 23-hydroxylation of
25-OH-D
or 1,25-(OH)
D
, although
this may be the result of the assay conditions used rather than the
fact that CYP24 is incapable of direct 23-hydroxylation of vitamin D
substrates. We observed here both the formation of 24-oxo metabolites
as well as 24-oxo-23-hydroxylated metabolites from side-chain-extended
analogs. Consistent with CYP24 being multifunctional and being
responsible for the enzyme activities observed in HPK1A-ras cells here, were the observations that: 1) the 23- and
24-hydroxylase activities are present together; 2) the activities are
both vitamin D-inducible; 3) and yet there appears only one mRNA
species in Northern blots from such vitamin D-inducible HPK1A-ras cell total RNA when hybridized with fragments or full-length
probes of CYP24
(
)suggesting that even if a
separate 23-hydroxylase exists it is only distantly related. Thus, in
the data reported here, it is not clear if the change in the ratio of
23:24-hydroxylation is a result of differences in the substrate
preferences of two different cytochromes P450 or is a result of
constraints of the substrate pocket of a single cytochrome with dual
properties, namely a CYP24 capable of both 23- and 24-hydroxylation.
Figure 9:
Model for the interaction of the
cytochromes P450, CYP27 and CYP24, with their respective substrates. A
two-chamber model is proposed which allows for accommodation of the
vitamin D nucleus as well as its side chain. The model for CYP27
comprises a large nonspecific pocket for different sterol/secosterol
ring structures and a more specific adjoining pocket for
cholesterol-type side chains of varying length. The model for CYP24
comprises a smaller, secosterol-specific binding pocket and a longer,
open-ended cylindrical chamber to accommodate side chains of varying
length. The relative positions of the heme group of each cytochrome is
illustrated by the porphyrin ring structure, each coordinating an iron
atom.
On
the other hand, the HPK1A-ras 23- and 24-hydroxylases (which
could be the same enzyme) are specific for vitamin D substrates, but
seem to ignore lengthening of the side chain, continuing to insert a
hydroxyl group at C-23 or C-24. Although the distance from the C-23 or
C-24 to the terminal carbons changes by as much as three carbons in our
experiments here, it is worth noting that the distance from C-23 and
C-24 to the vitamin D nucleus remains approximately the same. It is
thus attractive to hypothesize that the structural feature used by the
24- (and 23-) hydroxylase(s) to locate its hydroxylation site is the
distance between the ring structure (possibly the D ring because this
is closest) and the side chain carbon. Recently, Reddy et al.(32) reported the lack of 23-hydroxylation of analogs with
a 16-ene in the D ring of 1,25-(OH)D
implying
that the 23-hydroxylase activity is sensitive to such changes.
Similarly, we have also shown that another analog
20-epi-1,25-(OH)
D
is not readily
23-hydroxylated(31) . We conclude that the substrate binding
pocket of CYP24 must be cylindrical, permitting the entry of a specific
cholesterol-like side chain in addition to recognizing structural
features of the D ring and performing hydroxylation at a site (or
sites) part way down the cylinder. One would hypothesize that the
cylinder must be open at both ends in order to allow entry of the side
chain while continuing to facilitate hydroxylation of homologated
derivatives with up to three carbon atom extensions. These crude ideas
of the shapes of substrate binding pockets are certain to be
substantially refined in the foreseeable future as the deduced amino
acid structures of CYP24 and CYP27 are now available and recent
advances have occurred in the computerized molecular modelling of other
related steroidal cytochrome P450 isoforms (33) based upon x-ray
crystallographic data obtained from prokaryotic enzymes.
(
)The experiments involving transfection of cDNAs for
vitamin D-related cytochromes performed here is an extension of the
cultured cell technology and also allows for the dissection of the
cytochrome P450 isoforms involved in these hydroxylations in the
cultured cells. With the broadening of this approach to include
site-directed mutagenesis of the putative residues involved in the
substrate binding pockets, the models built from substrate studies
performed here can be tested and refined.
Table: 0p4in
HPLC conditions:
Zorbax-CN (0.46
25 cm) column eluted with HIM 91/7/2 at a flow
rate of 2 ml/min.
Table: 0p4in
HPLC conditions: Zorbax-CN (0.46
25
cm) column eluted with HIM 91/7/2 at a flow rate of 1 ml/min.
or
D
, vitamin D
or vitamin D
; OH or
(OH)
, hydroxy or dihydroxy, e.g. 1
,25-(OH)
D
=
1
,25-dihydroxyvitamin D
(note that in the nomenclature
used, no distinction between C-26 and C-27 is implied); BSA, bovine
serum albumin; DPPD, N,N`-diphenylethylenediamine;
HIM, hexane/isopropyl alcohol/methanol; RRT, relative retention time
(compared to 1
-OH-D
);
-MEM,
-minimal
essential medium; HPLC, high performance liquid chromatography.
25 µg) to allow direct comparison with the
chemically synthesized 24-hydroxy-MC1127, by one- and two-dimensional
proton NMR analysis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.