Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human CYP24A1

Article abstract of DOI:10.1016/j.jsbmb.2015.02.010

CYP11A1 hydroxylates vitamin D3 producing 20S-hydroxyvitamin D3 [20(OH)D3] and 20S,23-dihydroxyvitamin D3 [20,23(OH)₂D3] as the major and most characterized metabolites. Both display immuno-regulatory and anti-cancer properties while being non-calcemic. A previous study indicated 20(OH)D3 can be metabolized by rat CYP24A1 to products including 20S,24-dihydroxyvitamin D3 [20,24(OH)₂D3] and 20S,25-dihydroxyvitamin D3, with both producing greater inhibition of melanoma colony formation than 20(OH)D3. The aim of this study was to characterize the ability of rat and human CYP24A1 to metabolize 20(OH)D3 and 20,23(OH)₂D3. Both isoforms metabolized 20(OH)D3 to the same dihydroxyvitamin D species with no secondary metabolites being observed. Hydroxylation at C24 produced both enantiomers of 20,24(OH)₂D3. For rat CYP24A1 the preferred initial site of hydroxylation was at C24 whereas the human enzyme preferred C25. 20,23(OH)₂D3 was initially metabolized to 20S,23,24-trihydroxyvitamin D3 and 20S,23,25-trihydroxyvitamin D3 by rat and human CYP24A1 as determined by NMR, with both isoforms showing a preference for initial hydroxylation at C25. CYP24A1 was able to further oxidize these metabolites in a series of reactions which included the cleavage of C23-C24 bond, as indicated by high resolution mass spectrometry of the products, analogous to the catabolism of 1,25(OH)₂D3 via the C24-oxidation pathway. Similar catalytic efficiencies were observed for the metabolism of 20(OH)D3 and 20,23(OH)₂D3 by human CYP24A1 and were lower than for the metabolism of 1,25(OH)₂D3. We conclude that rat and human CYP24A1 metabolizes 20(OH)D3 producing only dihydroxyvitamin D3 species as products which retain biological activity, whereas 20,23(OH)₂D3 undergoes multiple oxidations which include cleavage of the side chain.

Full text of DOI:10.1016/j.jsbmb.2015.02.010

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SBMB 4375 1–13
Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry & Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
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Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin
D3 by rat and human CYP24A1
a
b
b,c
d
b
3
4
Q1
Q2
Elaine W. Tieu , Wei Li , Jianjun Chen , Tae-Kang Kim , Dejian Ma ,
d,e
a,
Andrzej T. Slominski , Robert C. Tuckey *
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, WA 6009, Australia
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN, USA
Department of Pharmaceutical Sciences, School of Pharmacy, South College, Knoxville, TN, USA
Department of Pathology and Laboratory Medicine, Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN, USA
Department of Medicine, Division of Rheumatology and Connective Tissue Diseases, University of Tennessee Health Science Center, Memphis, TN, USA
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6
7
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a
b
c
d
e
A R T I C L E I N F O
A B S T R A C T
Article history:
CYP11A1 hydroxylates vitamin D3 producing 20S-hydroxyvitamin D3 [20(OH)D3] and 20S,23-
Received 28 November 2014
Received in revised form 15 February 2015
Accepted 16 February 2015
Available online xxx
dihydroxyvitamin D3 [20,23(OH)
immuno-regulatory and anti-cancer properties while being non-calcemic. A previous study indicated 20
2
D3] as the major and most characterized metabolites. Both display
(
(
OH)D3 can be metabolized by rat CYP24A1 to products including 20S,24-dihydroxyvitamin D3 [20,24
OH) D3] and 20S,25-dihydroxyvitamin D3, with both producing greater inhibition of melanoma colony
2
formation than 20(OH)D3. The aim of this study was to characterize the ability of rat and human
CYP24A1 to metabolize 20(OH)D3 and 20,23(OH) D3. Both isoforms metabolized 20(OH)D3 to the same
dihydroxyvitamin species with no secondary metabolites being observed. Hydroxylation at
C24 produced both enantiomers of 20,24(OH) D3. For rat CYP24A1 the preferred initial site of
hydroxylation was at C24 whereas the human enzyme preferred C25. 20,23(OH) D3 was initially
Keywords:
2
2
2
0-Hydroxyvitamin D3
0,23-Dihydroxyvitamin D3
D
Cytochrome P450
CYP24A1
2
2
Vitamin D3
Phospholipid vesicles
metabolized to 20S,23,24-trihydroxyvitamin D3 and 20S,23,25-trihydroxyvitamin D3 by rat and human
CYP24A1 as determined by NMR, with both isoforms showing a preference for initial hydroxylation at
C25. CYP24A1 was able to further oxidize these metabolites in a series of reactions which included the
cleavage of C23-C24 bond, as indicated by high resolution mass spectrometry of the products, analogous
to the catabolism of 1,25(OH)
observed for the metabolism of 20(OH)D3 and 20,23(OH)
for the metabolism of 1,25(OH) D3. We conclude that rat and human CYP24A1 metabolizes 20(OH)
D3 producing only dihydroxyvitamin D3 species as products which retain biological activity, whereas
0,23(OH) D3 undergoes multiple oxidations which include cleavage of the side chain.
ã 2015 Elsevier Ltd. All rights reserved.
2
D3 via the C24-oxidation pathway. Similar catalytic efficiencies were
2
D3 by human CYP24A1 and were lower than
2
2
2
1
0
13
1
. Introduction
Inactivation of vitamin D by CYP24A1 can take place via two
catabolic pathways where initial hydroxylation occurs at either
C24 or C23, termed the C24-oxidation and C23-oxidation path-
1
4
1
1
1
2
15
16
CYP24A1 is the mitochondrial cytochrome P450 responsible for
the catabolism of 1 ,25-dihydroxyvitamin D3 [1,25(OH) D3].
a
2
ways, respectively [1,2]. The sequential oxidation of 1,25(OH)
the C24-oxidation pathway results in the formation of 24-oxo-
,23,25-trihydroxyvitamin D3 which undergoes side chain cleav-
2
D3 in
1
1
1
2
7
8
9
0
1
Abbreviations: 1,25(OH)
hydroxyvitamin D3; 20(OH)D3, 20-hydroxyvitamin D3; 20,23(OH)
dihydroxyvitamin D3; 20,23,24(OH) D3, 20,23,24-trihydroxyvitamin D3; 20,23,25
OH) D3, 20,23,25-trihydroxyvitamin D3; cyclodextrin, 2-hydroxylpropyl- -cyclo-
dextrin; TOCSY, H- H total correlation spectroscopy; COSY, H- H correlation
2
D3,
1
a
,25-dihydroxyvitamin D3; 25(OH)D3, 25-
age between C23 and C24 with the final product, calcitroic acid,
being excreted. The C23-oxidation pathway produces 1,25-
dihydroxyvitamin D3-26,23-lactone. There are species differences
in the preference for these pathways, with rat CYP24A1 favoring
the C24-oxidation pathway [3–5] and human CYP24A1 exhibiting
both pathways [1,3,6,7].
It has been established in the last decade that CYP11A1 (also
known as cytochrome P450scc) can metabolize vitamin D3 to
produce many novel mono- and poly-hydroxyvitamin D metab-
olites, the major ones being 20S-hydroxyvitamin D3 [20(OH)D3]
2
D3, 20,23-
3
21
(
3
b
2
2
2
3
1
1
1
1
1
13
spectroscopy; HSQC, H- C heteronuclear single quantum correlation spectrosco-
py; HMBC, H-
phospholipid.
1
13
Q3 24
25
C heteronuclear multiple bond correlation spectroscopy; PL,
*
Corresponding author at: School of Chemistry and Biochemistry, The University
26
of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
Tel.: +61 8 6488 3040; fax: +61 8 6488 1148.
2
2
7
8
E-mail address: robert.tuckey@uwa.edu.au (R.C. Tuckey).
http://dx.doi.org/10.1016/j.jsbmb.2015.02.010
0
960-0760/ã 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: E.W. Tieu, et al., Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human
CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010

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91
and 20S,23-dihydroxyvitamin D3 [20,23(OH)
2
D3] [8–18]. This
2.2. Preparation of enzymes
pathway was initially elucidated from in vitro studies with purified
bovine CYP11A1, and more recently has been demonstrated to
occur in keratinocyte cell cultures and in fragments of adrenal
glands and human placenta incubated with vitamin D3 [16,18,19].
92
93
Rat and human CYP24A1 were expressed and purified as
previously described [33,35]. Human and mouse adrenodoxin, and
human adrenodoxin reductase were expressed in Escherichia coli
and purified as before [17,36,37].
Q5 94
95
Most recently, 20(OH)D3 and 20,23(OH)
relative levels similar to the classical 25(OH)D3 and 1,25(OH)
2
D3 have been detected at
D3 in
2
96
97
human epidermal tissue [20], confirming their production in vivo.
Possible physiological roles for these metabolites remain to be
established.
2.3. Measurement of secosteroid metabolism by CYP24A1 in a
phospholipid vesicle reconstituted system
98
99
2
0(OH)D3 and 20,23(OH)
2
D3 are the most extensively studied
Dioleoyl phosphaditycholine (1.08
mmol), bovine heart cardi-
of the CYP11A1-derived secosteroids in terms of their in vitro
biological actions. Both act as biased agonists on the vitamin D
receptor and thus display many, but not all, of the biological effects
olipin (0.19 mol) and the secosteroid substrate (as required) were
m
100
aliquotted into glass tubes and the ethanol solvent removed under
nitrogen gas. Assay buffer, pH 7.4 (20 mM HEPES, 100 mM NaCl,
0.1 mM EDTA and 0.1 mM DTT) (0.5 mL) was added to the dried
lipid mixture. This was purged for 30 s with nitrogen gas and then
tubes sonicated for approximately 10 min in a bath-type sonicator,
until the solution was clear [38]. The incubation mixture was
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
of 1,25(OH)
agonists on ROR
2
D3 [16,21–23]. They can both also act as inverse
and ROR [24]. They have the ability to promote
a
g
differentiation and suppress proliferation of a number of normal
and cancerous cells in vitro, such as keratinocytes, melanocytes,
fibroblasts, melanoma and leukemia cells [16,21–23,25–28]. 20
composed of vesicles (510
for human CYP24A1, and 0.05–1
mouse adrenodoxin (15 M), human adrenodoxin reductase
(0.4 M), glucose-6-phosphate (2 mM), glucose-6-phosphate de-
hydrogenase (2 U/mL) and NADPH (50 M), in assay buffer.
m
M phospholipid), P450 (0.01–0.05
mM
(
OH)D3 and 20,23(OH)
normal and immortalized keratinocytes by increasing the expres-
sion of I B, thus attenuating the transcriptional activity of NF-
23,27,29]. In addition, both 20(OH)D3 and 20,23(OH) D3 possess
2
D3 promote anti-inflammatory activity in
m
M for rat CYP24A1), human or
m
k
k
B
m
[
2
m
anti-fibrotic properties on human dermal fibroblasts isolated from
scleroderma and normal subjects [26]. In rodent models,
administration of 20(OH)D3 has been found to be effective in
reducing the symptoms of scleroderma [26] and rheumatoid
arthritis [16] as well as protecting DNA in skin from damage caused
Following preincubation for 3 min, reactions were started by the
addition of adrenodoxin and samples (0.25–2.5 mL) incubated at
ꢀ
37 C, with shaking (see for reaction times). Reactions were
terminated by the addition of 2.5-volumes of ice-cold dichloro-
methane and samples were extracted four times with vortexing
and centrifugation. The samples were dried under nitrogen gas and
redissolved in ethanol for HPLC analysis. The samples were
analysed on a PerkinElmer modular HPLC system which comprised
a Biocompatible Binary LC pump (model 250; PerkinElmer
Corporation, MA, USA) and a UV detector (LC-135C; PerkinElmer
by UV irradiation [30]. Importantly, unlike 1,25(OH)
OH)D3 and 20,23(OH) D3 are non-calcemic in rodents at high
concentrations [25,26,31]. Thus both 20(OH)D3 and 20,23
OH) D3 have therapeutic potential for the treatment of hyper-
2
D3, both 20
(
2
(
2
proliferative and inflammatory disorders.
Recently it has been reported that 20(OH)D3 can be further
metabolized by cytochromes P450 involved in the metabolism of
vitamin D3. Human CYP27A1 converts 20(OH)D3 to 20S,25-
Corporation, MA, USA) set at 265 nm, equipped with
C18 analytical column (Grace Alltima, 250 Â 4.6 mm, particle size
m; Grace Davison Discovery Sciences, VIC, Australia). Different
HPLC programs were used depending on the substrate. For the
separation of monohydroxyvitamin substrates and their
a
5
m
dihydroxyvitamin D3 [20,25(OH)
min D3 [20,26(OH) D3], whereas rat CYP24A1 produces 20S,24-
dihydroxyvitamin D3 [20,24(OH) D3] and 20,25(OH) D3 [32,33].
Other P450 isoforms found in mouse liver microsomes also
produce 20,25(OH) D3 and 20,26(OH) D3 [34]. These resulting
2
D3] and 20S,26-dihydroxyvita-
2
D
2
2
products, a 20 min gradient from 45% (v/v) acetonitrile in water
to 100% acetonitrile, followed by 30 min at 100% acetonitrile, all at
a flow rate of 0.5 mL/min (HPLC Program A), was used. A 40 min
gradient from 30% (v/v) acetonitrile in water to 100% acetonitrile,
followed by 15 min at 100% acetonitrile, all at a flow rate of 0.5 mL/
min, was used to separate polyhydroxyvitamin D substrates and
products (HPLC Program B). The peak areas were integrated using
Clarity software (DataApex, Prague, Czech Republic). Kinetic
parameters were determined by fitting the Michaelis–Menten
equation to the experimental data using Kaleidagraph, version 4.1
(Synergy Software, Reading, PA, U.S.A.).
2
2
secosteroids are more potent than the parent 20(OH)D3 in the
inhibition of melanoma colony formation [33]. However, addition
of the 1
,20S-dihydroxyvitamin D3, confers some calcemic activity
although less than that observed with 1,25(OH) D3 [25]. Recently
we have successfully extracted and partially purified human
CYP24A1, and characterized its activity toward 1,25(OH) D3 and
a-hydroxyl group to 20(OH)D3 by CYP27B1 producing
1
a
2
2
the intermediates of the C24-oxidation pathway [35]. In the
present study we used human CYP24A1, along with rat CYP24A1, to
characterize the metabolism of both 20(OH)D3 and 20,23(OH)
137
2
D3.
2
2.4. Enzymatic synthesis and HPLC purification of 20,24(OH) D3
8
8
0
1
138
2
. Materials and methods
To produce 20,24(OH)
incubation (30 mL) of rat CYP24A1 with 20(OH)D3 was carried out,
as described previously [33]. The 20,24(OH) D3 and other products
2
D3 for NMR analysis, a large scale
1
39
140
41
2.1. Materials
2
1
were purified by HPLC, as outlined before [33], using a Grace
Alltima column (as above) and a 45–58% (v/v) acetonitrile in water
gradient over 25 min followed by a 10 min gradient from 58% (v/v)
acetonitrile in water to 100% acetonitrile, ending with 20 min at
100% acetonitrile, all at a flow rate of 0.5 mL/min (HPLC Program C).
A further HPLC purification step was carried out using the same
column employing a 45 min gradient from 64% (v/v) methanol in
water to 100% methanol, followed with 15 min at 100% methanol,
all at a flow rate of 0.5 mL/min. Collected products were pooled and
dried under nitrogen, dissolved in ethanol and the amount of
8
8
8
8
8
8
8
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9
2
3
4
5
6
7
8
9
0
142
143
144
145
146
147
148
149
150
2
2
0(OH)D3 and 20,23(OH) D3 were synthesized from vitamin
D3 enzymatically using bovine CYP11A1 and were purified by TLC
and HPLC, as previously described [10,17]. Vitamin D3, dioleoyl
phosphaditylcholine, bovine heart cardiolipin, 2-hydroxylpropyl-
Q4
b
-cyclodextrin (cyclodextrin) and glucose-6-phosphate were
purchased from Sigma (Sydney, Australia). Glucose-6-phosphate
dehydrogenase was from Roche (Mannheim, Germany). All
solvents were of HPLC grade and were purchased from Merck
(
Darmstadt, Germany).
Please cite this article in press as: E.W. Tieu, et al., Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human
CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010

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E.W. Tieu et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx
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1
1
51
52
211
12
purified secosteroid was measured spectrophotometrically using
an extinction coefficient of 18,000 M cm [39].
3. Results
À1
À1
2
3
.1. Rat CYP24A1 metabolizes 20(OH)D3 to produce the two
1
53
213
2
.5. Large scale enzymatic synthesis of metabolites of 20,23(OH) D3
2
C24 enantiomers of 20,24-dihydroxyvitamin D3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
A stock solution of 20,23(OH)
prepared by drying an aliquot of 20,23(OH)
in 4.5% (w/v) cyclodextrin by stirring in the dark overnight.
Expressed rat CYP24A1 (1 M) was incubated with the 20,23
M) at a final cyclodextrin concentration of 0.56%, for
2
D3 (0.45 mM) in cyclodextrin was
We have previously reported that rat CYP24A1 can metabolize
20(OH)D3 to at least five products, with the major two products
being identified by NMR as 20,24(OH) D3 and 20,25(OH) D3 [33].
2 2
The three other products were characterized as species of
dihydroxyvitamin D3 by mass spectrometry, with the site of the
CYP24A1-catalysed hydroxylation unknown [33]. In this study, we
used enzymatic synthesis to produce enough of the third major
2
D3 and redissolving it
m
(
OH)
2
D3 (56
m
ꢀ
90 min at 37 C, in a 20 mL incubation. Other reaction components,
except phospholipids, were as described above for the phospho-
lipid vesicle system. The extraction of the products was also as
described above. Initial HPLC purification of the products was
carried out using HPLC Program B. One of the products, 20,23,25
product, Product C, (Fig.1) for NMR analysis. Product C (53
m
g) was
in 0.29% (w/v)
M rat CYP24A1 (see
produced by incubating 20(OH)D3 (53
cyclodextrin) in a 30 mL incubation with 1
mM
m
(
OH)
3
D3, required further purification which was done using a
Section 2.4), and purified by reverse phase HPLC using both
acetonitrile–water (Fig. 1) and methanol–water solvent systems.
The mass spectrum confirmed it was a dihydroxyvitamin D3 with
40 min gradient of 64% (v/v) methanol in water to 100% methanol,
followed by 20 min at 100% methanol on the same C18 column. The
yield of products formed was determined spectrophotometrically,
as described before.
+
the observed molecular ion m/z = 439.3188 [M + Na] , as reported
before [33] (Fig. S1).
2
29
230
31
The site of hydroxylation on 20(OH)D3 in Product C was
unambiguously assigned to be at the 24-position based on the
NMR spectra for this metabolite. First, none of the four methyl
1
69
2.6. Mass spectrometry
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
1
232
233
234
235
236
237
238
239
240
241
The molecular masses of the intermediates of the
carbons (C18, C21, C26, C27) are hydroxylated based on H NMR
1
13
CYP24A1 action on 20,23(OH)
2
D3 (low resolution mass spectra)
(Fig. 2A). H- C HSQC revealed the presence of a new methine
13
1
1
were determined by 2 dimensional (2D) UPLC tandem mass
spectrometry in a similar manner to that described in detail by
Clarke et al. [40]. The system consisted of two Agilent 1290 UPLC
binary pumps coupled to an Agilent 6460 triple quadrupole
tandem mass spectrometer with a Jetstream source. Separation of
the intermediates was carried out by two pentafluorophenyl (PFP)
columns (100 Å), both run isocratically with 80% (v/v) methanol in
water containing 0.1% (v/v) formic acid. The mass spectrometer
was operated in positive ESI (electrospray ionization) mode. Three
group at 3.22 ppm ( C at 78.3 ppm, Fig. 2B). H- H TOCSY (Fig. 2C)
clearly showed that this methine is in the same spin system as 26/
1
27-CH
3
( H at 0.91 ppm), indicating the hydroxylation occurred in
1
1
the side chain. From the H- H COSY (Fig. 2D) spectrum, this
1
1
methine ( H at 3.22 ppm) showed a strong correlation to 25-CH ( H
1
1
13
at 1.62 ppm) and 23-CH
HMBC (Fig. 2E), 26/27-CH
correlation to the new methine ( C at 78.3 ppm), in addition to the
2
( H at 1.62 and 1.45 ppm). From H- C
1
3
( H at 0.91 ppm) showed a strong
13
m
g of each was diluted 1/1000 with 70% (v/v) methanol in water
and 20 L was injected to the system.
m
High resolution mass spectra were acquired in a Waters Xevo
G2-S system (Waters, Milford, MA) utilizing an ESI source with a
Waters Acquity I-Class UPLC and BEH C18 column (2.1 mm
 50 mm, 1.7
mm, Waters, Milford, USA). Data were collected and
processed by Masslynx 4.1 software.
1
88
2.7. NMR
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07 Q6
08
09
10
NMR measurements of 20,24(OH)
inverse triple-resonance 3 mm probe on a Varian Unity Inova
00 MHz spectrometer (Agilent Technologies, Inc., Santa Clara, CA,
USA). Sample was dissolved in CD OD and transferred to a 3 mm
2
D3 were performed using an
5
3
Shigemi NMR tube (Shigemi Inc., Allison Park, PA, USA). Tempera-
ꢀ
ture was regulated at 22 C and was controlled with an accuracy of
ꢀ
Æ0.1 C. Chemical shifts were referenced to residual solvent peaks
3
for CD OD (3.31 ppm for proton and 49.15 ppm for carbon).
1
1
Standard two-dimensional NMR experiments [ H- H total corre-
1
1
lation spectroscopy (TOCSY, mixing time = 80 ms), H- H correla-
tion spectroscopy (COSY), 1H- C heteronuclear single quantum
13
1
13
correlation spectroscopy (HSQC), and H- C heteronuclear multi-
ple bond correlation spectroscopy (HMBC)] were acquired in order
to fully elucidate the structures of the metabolites. All data were
processed using ACD software (Advanced Chemistry Development,
Toronto, ON, Canada), with zero-filling in the direct dimension and
linear prediction in the indirect dimension. NMR data of 20,23,25
(
OH)
Bruker Avance III 400 MHz, with a BBO 5 mm probe with z-gradient
Bruker BioSpin, Billerica, MA). The spectrometer was equipped
3
D3 and 20,23,24(OH)
3
D3 were acquired in CDCl
3
, using
Fig. 1. 20-Hydroxyvitamin D3 is metabolized by rat CYP24A1. Rat CYP24A1 (1 mM)
was incubated with 20(OH)D3 dissolved in 0.45% (w/v) cyclodextrin, for 90 min at
ꢀ
3
7 C in a reconstituted system containing adrenodoxin and adrenodoxin reductase.
(
The reaction mixture was analysed using HPLC Program C (see Section 2.4). (A)
Chromatogram showing test reaction. (B) Chromatogram showing control reaction
with NADPH omitted.
with an autosampler and IconNMR Automation was used within
TopSpin 3.0 for data acquisition.
Please cite this article in press as: E.W. Tieu, et al., Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human
CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010

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Fig. 2. NMR reveals that Product C is 20,24-dihydroxyvitamin D3. (A) 1D Proton; (B) 1H-13C HSQC; (C) 1H- H TOCSY; (D) H- H COSY; (E) 1H-13C HMBC.
1
1
1
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CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010

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5
2
2
2
2
2
2
42
43
44
45
46
47
expected correlation to 25-CH (13C at 34.8 ppm). Taken together,
the above analysis shows that the hydroxylation site can be
unambiguously assigned to the 24-position. This product therefore
represents the other C24 enantiomer of the previously character-
248
249
250
251
252
253
summarized in Supplementary Table 1. For comparison, we also
included the assignments for the parent compound 20(OH)D3 and
the other enantiomer, Product B [33]. However, we were unable to
establish the absolute configurations at the 24-position for both
isomers at this stage because of the lack of high resolution NMR
data, due to the limiting amount of these secosteroids.
ized major reaction product, 20,24(OH)
2
D3 (Fig. 1), originally
designated as Product B [33]. The full assignments for Product C are
Fig. 3. Human CYP24A1 acts on 20-hydroxyvitamin D3 producing similar products to the rat enzyme. (A) 20(OH)D3 was incorporated in phospholipid vesicles at a ratio of
ꢀ
0
.05 mol/mol phospholipid and incubated with human CYP24A1 (0.05
m
M) in a reconstituted system containing adrenodoxin and adrenodoxin reductase, for 30 min at 37 C.
Samples were analysed using HPLC Program A (see Section 2.3). Inset, enlarged view of the chromatogram from 24 to 34 min. (B) Control reaction showing that lack of
products when adrenodoxin was omitted. Peaks present in the control as well as the test at retention times of 18, 20 and 34 min, which appear to originate from the
phospholipids, were not considered to be 20(OH)D3 products. (C) Time course for the metabolism of 20(OH)D3 in phospholipid vesicles by human CYP24A1. The same
conditions were used as outlined in Fig. 3A.
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CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010

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2
2
54
55
264
265
3
.2. Human CYP24A1 can metabolize 20(OH)D3 to dihydroxyvitamin
The two major ones were identified as 20,25(OH)
and 20,24(OH) D3 (Product B) by comparison of their retention
times to those of the authentic standards produced using rat
CYP24A1 [33]. The other enantiomer of 20,24(OH) D3 (Product C)
seen with the rat enzyme (Fig.1) was present as a minor product of
the human enzyme, and a peak with a shoulder corresponding to
Products D and E was also seen (Fig. 3).
2
D3 (Product A)
D products similar to rat CYP24A1
2
2
66
2
2
2
2
2
2
2
2
56
57
58
59
60
61
62
63
267
268
269
270
271
272
273
274
The bacterial expression and partial purification of human
CYP24A1 [35] enabled us to test this isoform of the enzyme on the
metabolism of 20(OH)D3, although only at a low enzyme
concentration due to its low expression. As for the rat enzyme
2
[
33], 20(OH)D3 was incorporated into phospholipid vesicles, a
system that mimics the inner mitochondrial membrane where the
enzyme is located in vivo [38,41–43]. Human
CYP24A1 metabolized 20(OH)D3 to several products (Fig. 3).
The time course for metabolism of 20(OH)D3 by human
ꢀ
CYP24A1 is shown in Fig. 3C. After the 1 h incubation at 37 C with
0.05
mM P450,18% of the 20(OH)D3 substrate was consumed. None
of the products measured in the time course displayed a lag,
Fig. 4. Both rat and human CYP24A1 metabolize 20,23-dihydroxyvitamin D3. (A) Rat CYP24A1 (1
m
M) was incubated with 20,23(OH)
D3 incorporated in phospholipid vesicles, at a molar ratio of 0.05 mol/mol
M) was incubated with 20,23(OH) D3 incorporated in
D3 (E and F)
M human CYP24A1 (F
D3 (not shown) showed the
2
D3 (50 mM) dissolved in 0.45% (w/v)
ꢀ
cyclodextrin, for 1 h at 37 C. (B, C) Rat CYP24A1 (1
phospholipid, for 1 h at 37 C in the absence (B) or presence (C) of adrenodoxin. (D) Human CYP24A1 (0.05
m
M) was incubated with 20,23(OH)
2
ꢀ
m
2
phospholipid vesicles (0.05 mol/mol), under the same conditions as described for C; inset, expanded view of the chromatogram from 34 to 46 min. 20,23,25(OH)
3
or 20,23,24(OH)
3
D3 (G and H) were incorporated into phospholipid vesicles (0.05 mol/mol), and incubated with 1
m
M rat CYP24A1 (E and G) or 0.05
m
ꢀ
and H) at 37 C for 30 min. Samples were analysed by HPLC using Program B (see Section 2.3). The controls for 20,23,24(OH)
3
D3 and 20,23,25(OH)
3
2
same contaminant peak as seen in Fig. 4B. The asterisks denotes the putative pre-20,23(OH) D3.
Please cite this article in press as: E.W. Tieu, et al., Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human
CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010

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7
Table 1
Q10
Mass spectrometric identification of products formed from metabolism of 20,23-dihydroxyvitamin D3 by CYP24A1. Products 1, 3 and the combined product peak for 5 A and B
were formed from the action of rat CYP24A1 on 20,23(OH)
2
D3. Products 5A and 2 were made from 20,23,25(OH)
3
D3. Products 5B and 6 were made from 20,23,24(OH)
3
D3.
Product
1
Exact mass
455.3151
Calc. mass
Error (ppm)
Formula
Observed
Assignment
455.3139
415.3214
397.3108
379.3002
2.6
1.9
3
C
C
C
C
27
H
27
H
27
H
27
H
44
O
O
O
4
3
2
Na
Na
M + Na+
20,23,24(OH)
3
D3
D3
+
415.3222
43
M + H –H
2
O
+
3
3
97.312
79.301
41
M + H –2H
2
O
O
+
2.1
39
O
M + H –3H
2
3
455.3138
15.3205
455.3139
415.3214
397.3108
379.3002
0.2
2.2
C
C
C
C
27
H
27
H
27
H
27
H
44
O
O
O
4
3
2
M + Na+
20,23,25(OH)
3
+
4
43
M + H –H
2
O
+
3
3
97.3099
79.2997
À2.3
41
M + H –2H
2
O
O
+
À1.3
39
O
M + H –3H
2
5
5
5
A + 5B
469.2914
29.3006
11.2901
469.293
429.3005
411.2899
À3.4
0.2
0.5
C
C
C
27
27
27
H
H
H
42
O
5
Na
Na
Na
M + Na+
4
20(OH)D3-23COOH and Dehydro-20,23,24,Y(OH) D3
+
4
4
41
O
4
M + H –H
2
O
+
39
O
3
2
M + H –2H O
A
B
483.2719
57.2429
39.2329
483.2723
357.243
339.2324
À0.8
À0.3
1.5
C
C
C
27
H
23
H
23
H
40
33
31
O
O
6
?
Unknown
20(OH)D3-23COOH
+
3
3
M + H –H
M + H –2H O
2
2
O
+
3
O
2
469.2921
469.293
À1.9
À0.9
À0.5
2.3
C
C
C
C
27
H
27
H
27
H
27
H
42
O
5
4
M + Na+
Dehydro-20,23,24,Y(OH)
4
D3
D3
+
4
29.3001
429.3005
411.2899
393.2794
41
O
M + H –H
2
O
+
411.2897
39
O
3
M + H –2H
2
O
O
+
3
93.2803
37
O
2
M + H –3H
2
6
469.2934
11.2901
93.2804
469.293
411.2899
393.2794
0.9
0.5
2.5
C
C
C
27
H
27
H
27
H
42
39
37
O
O
5
3
Na
M + Na+
Dehydro-20,23,24,X(OH)
4
+
4
M + H –2H
2
2
O
O
+
3
O
2
M + H –3H
2
2
2
2
2
2
75
76
77
78
79
80
311
312
313
314
315
316
consistent with all of them being primary products with the
addition of just one hydroxyl group, as reported for the rat enzyme
2
that of the substrate, 20,23(OH) D3 (denoted by the asterisk in
Fig. 4D). This peak was also present in the control incubation where
adrenodoxin was omitted indicating that CYP24A1 is not involved
in its formation (Fig. 4B). This peak was collected and shown to be a
vitamin D3 derivative, with the same UV spectrum as 20,23
(OH) D3 (not shown). Rechromatography of the collected product
2
in the same solvent system revealed that it was partially converted
back to a compound with the same retention time as 20,23
[33]. The time course also shows that human CYP24A1 favors
hydroxylating at the C25 position of 20(OH)D3 over the
C24 position at an approximate ratio of 3:1, the opposite of what
is observed for the rat enzyme (Fig. 1 and [33]).
3
17
2
2
81
82
318
319
3.3. Metabolism of 20,23(OH)
2
D3 by CYP24A1 results in some cleavage
of the side chain
(OH)
2
D3. It would therefore appear to be pre-20,23(OH)
2
D3, which
D3
3
20
results from the reversible thermoisomerization of 20,23(OH)
[44].
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
2
Initially, the metabolism of 20,23(OH) D3 by rat CYP24A1 was
measured with substrate dissolved in cyclodextrin as this system is
good for scaling reactions up to produce sufficient vitamin D
metabolites for NMR. It enables high substrate concentrations to be
used and has been employed extensively by us in the past for
synthesizing products of other vitamin D-metabolizing enzymes
2
The products of 20,23(OH) D3 metabolism by rat CYP24A1 with
the substrate dissolved in cyclodextrin (Fig. 4A) were collected and
analysed by mass spectrometry. High resolution mass spectrome-
try of Product 1 gave the parent ion at m/z = 445.3138 [M + Na]+
corresponding to a molecular weight of 432.6359, indicating that it
is a trihydroxyvitamin D3 species (Table 1, Fig. S2A). Product 3 gave
[10,12,33]. Three major products were observed in a 1 h incubation
+
of rat CYP24A1 using this system (Fig. 4A). The two major ones
were identified as 20S,23,24-trihydroxyvitamin D3 [20,23,24
the parent ion at m/z = 455.3137 [M + Na] , indicating that it is also
a trihydroxyvitamin D3 species (Table 1, Fig. S2B). Analysis of
Product 2 by low resolution mass spectrometry gave the parent ion
(
(
OH)
OH)
3
D3] and 20S,23,25-trihydroxyvitamin D3 [20,23,25
+
3
D3] by NMR, as presented in detail later. In contrast to
with m/z = 453.1 [M + Na] and additional ions with m/z = 413.1 and
+
+
cyclodextrin, incubation of rat CYP24A1 with 20,23
OH) D3 incorporated into phospholipid vesicles resulted in the
2 2
395.1, which correspond to [M + H–H O] and [M + H–2H O] ,
(
2
respectively. The molecular weight of 430.1 suggests that this
product is formed by oxidation of one of the three side chain
hydroxyl groups in Product 1 or Product 3 to a ketone (producing a
dehydro-trihydroxyvitamin D3 species), an oxidation known to
appearance of several more products (Fig. 4C), which are likely to
be downstream metabolites arising from the major products
observed in Fig. 4A. This shows that the cyclodextrin reconstituted
system is not conducive to the conversion of primary products to
downstream secondary products. The same primary products
occur for the 24-hydroxyl group of 1,24,25(OH)
(OH) D3 in the C24-oxidation pathway of vitamin D inactivation
[35,45]. The broad peak labeled as Product 5A and B (Fig. 4C),
produced from 20,23(OH) D3 in phospholipid vesicles, was a
3
D3 and 24,25
2
(
Products 1–3) were seen for the human enzyme based on their
identical retention times to the products from the rat enzyme,
although they were produced in different proportions (Fig. 4D).
Due to its low expression and lability, only a low concentration of
the human enzyme was used in this experiment which was carried
2
mixture of two secosteroids based on its retention time and mass
spectrum, and will be described later.
343
344
2
out with 20,23(OH) D3 incorporated into phospholipid vesicles.
3.4. Enzymatic production of 20,23,24(OH)
3
D3, and 20,23,25
An additional major product (Product 4) was seen for the human
enzyme, but was only present in a trace amount for rat CYP24A1.
(OH) D3 by CYP24A1 for structure determination
3
345
346
2
In incubations of 20,23(OH) D3 with CYP24A1, a small peak was
As seen in Fig. 4A, rat CYP24A1 almost completely metabolized
20,23(OH) D3 solubilized in cyclodextrin and converted it to two
consistently detected with a retention time slightly longer than
2
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CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010

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Fig. 5. NMR reveals that Product 3 is 20,23,25-trihydroxyvitamin D3. (A) 1D Proton; (B) 1H–13C HMBC; (C) 1H–13C HSQC. Peaks labeled with asterisk (*) sign were not
identified.
3
3
3
3
3
3
3
3
47
48
49
50
51
52
53
54
directly attached to it based on the 1H-13C HSQC (Fig. 5C). The
chemical shift of C24 of Product 3 is at 47.7 ppm and it is a
362
363
364
365
366
367
368
369
370
371
372
major trihydroxyvitamin D3 species, labeled as Product 1 and
Product 3. This reaction was scaled up to 20 mL to produce enough
of Products 1 and 3 to enable their structure to be determination by
1
13
methylene group (—CH
Based on the above analysis, Product 3 was assigned 20,23,25
(OH) D3.
3
Product 1 was positively identified as 20,23,24(OH) D3 (Fig. 6).
2
) as revealed by H- C HSQC data (Fig. 5C).
NMR. Almost complete conversion of 20,23(OH)
with 1 M rat CYP24A1 in a 90 min incubation. Overall, 83
Product 1 and 108 g of Product 3 were obtained following their
2
D3 was achieved
m
m
g of
3
m
purification in acetonitrile–water and methanol–water solvent
systems, which was sufficient for structure determination by NMR.
There are two sets of peaks centered at 3.48 ppm, and 3.30 ppm on
the proton spectrum of Product 1 (Fig. 6A), which are not present in
1
1
Product 3. Based on H- H TOCSY (Fig. 6B), both signals at
3.48 ppm, and 3.30 ppm shared the same spin system as 26/27-CH
(0.83/0.79 ppm). They also showed correlations to 22-CH , and 21-
CH (Fig. 6B). By lowering the contour levels, weak correlations to
3
3
55
56
3
.5. NMR identification of 20,23,24(OH)
3
D3 and 20,23,25(OH)
D3
3
D3 as
3
major products of rat CYP24A1 action on 20,23(OH)
2
2
3
73
3
3
3
3
3
3
57
58
59
60
61
374
375
376
377
378
Based on the current NMR spectra and previous data for 20,23
OH) D3 [17], the site of the third hydroxylation of Product 3 was
assigned to be at the 25-position (Fig. 5A). The carbon chemical
shift of C25 moved from 25.2 ppm in 20,23(OH) D3 to 70.7 ppm in
Product 3 [17] (Fig. 5B). This carbon does not have any proton
23-CH were also positively identified (Fig. 6B). In addition, they
showed correlation to each other in COSY indicating that they
should be next to each other in the structure (Fig. 6C). Based on the
above analysis, they were assigned to 24-CH (3.30 ppm) and 25-CH
(
2
2
3
(3.48 ppm), and Product 1 was assigned 20,23,24(OH) D3. The full
Please cite this article in press as: E.W. Tieu, et al., Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human
CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010

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9
Fig. 6. Product 1 was identified as 20,23,24-trihydroxyvitamin D3 by NMR. (A) 1D Proton; (B) 1H– H TOCSY; (C) H– H COSY. Peaks labeled with asterisk (*) sign were not
1
1
1
identified.
3
3
79
80
395
396
assignments for 20,23,24(OH)
3
D3 (Product 1) and 20,23,25
1 h incubation with 1 mM rat CYP24A1, only 15% of substrate
(
OH)
3
D3 (Product 3) are summarized in Supplementary Table 2.
remained. The proportions of the two major products diminished
after the initial 10 min, suggesting that they serve as precursors
that give rise to some of the minor products (Fig. 7B). Consistent
with this, Products 2, 5A, 5B and 6 displayed lags in their time
courses suggesting that they are downstream secondary metab-
3
97
398
99
3
81
2
3.6. Time course for the metabolism of 20,23(OH) D3 by CYP24A1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
82
83
84
85
86
87
88
89
90
91
92
93
94
400
401
402
403
404
405
406
The time course for metabolism of 20,23(OH)
into phospholipid vesicles by human CYP24A1 shows that 20,23,25
OH) D3 was the major product of the reaction throughout the 1 h
incubation (Fig. 7A). The two minor products, 20,23,24(OH) D3 and
2
D3 incorporated
olites, arising from further metabolism of 20,23,24(OH)
20,23,25(OH) D3. Both rat and human CYP24A1 displayed a
preference for hydroxylating at C25 of 20,23(OH) D3, with the
proportion of 20,23,25(OH) D3 to 20,23,24(OH) D3 being
3
D3 or
(
3
3
3
2
Product 2, were not detected until 30 min of incubation but this was
likely due to the sensitivity of detection rather than there being a lag
in their production, especially for the primary product, 20,23,24
3
3
2.3:1 and 19:1 for the rat and human CYP24A1, respectively, at
the end of the incubation.
(
OH)
substrate was consumed in 1 h with 0.05
The time course for the metabolism of 20,23
OH) D3 incorporated into phospholipid vesicles by rat CYP24A1
Fig. 7B) shows that there is immediate formation of 20,23,24
OH) D3 (Product 1) and 20,23,25(OH) D3 (Product 3). After the
3
D3. The reaction was linear for the first 2 min and 18% of
407
408
m
M human CYP24A1.
3 3
3.7. 20,23,24(OH) D3, and 20,23,25(OH) D3 can be further
metabolized by CYP24A1
(
(
(
2
409
410
3 3
Following NMR, 20,23,24(OH) D3 and 20,23,25(OH) D3 were
repurified and used as substrates for CYP24A1. Due to the higher
3
3
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CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010

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E.W. Tieu et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx
4
4
4
4
4
4
4
4
4
4
4
4
43
44
45
46
47
48
49
50
51
52
53
54
5
B and accounted for 66% of the total products. Product 6 was
labeled according to its identical retention time to Product
6
(
produced from the action of CYP24A1 on 20,23(OH)
Fig. 4C) and was classified as a dehydro-tetrahydroxyvitamin
D3 species by high resolution mass spectrometry (Table 1, Fig. S4).
Since it is made from 20,23,24(OH) D3, it can be further classified
as dehydro-20,23,24,X(OH) D3 where both the position (X) of the
2
D3
3
4
new hydroxylation and which hydroxyl group is reduced to a
ketone are unknown. Two peaks with shorter retention times than
Product 5B (20 and 22 min, Fig. 4G) were observed and may
represent further downstream metabolites.
Like Product 6, Product 5B gave the parent with m/z = 469.2921
+
455
456
457
[M + Na] , as well as other ion fragments with m/z = 429.3001,
+
4
11.2897 and 393.2803, which correspond to [M + H–H
2
O] ,
O] , respectively (Table 1, Fig. S5).
This indicates that Product 5B has an exact mass of 446.3032 and is
also dehydro-tetrahydroxyvitamin D3 species (dehydro-
0,23,24,Y(OH) D3). The high resolution mass spectrum of the
+
+
2 2
[M + H–2H O] and [M + H–3H
4
4
4
4
4
4
4
4
4
4
58
59
60
61
62
63
64
65
66
67
a
2
4
combined peak labelled as Product 5A and 5B in Fig. 4C showed
ions with m/z values seen in the mass spectrum of Product 5A
produced from 20,23,25(OH)
mass spectrum of Product 5B produced from 20,23,24(OH)
from the peak in Fig. 4G) (Fig. S6). These two products have almost
identical retention times thus both products run together in the
reaction starting from 20,23(OH) D3 (Fig. 4C).
3
D3 (from the peak in Fig. 4E) and the
3
D3
(
2
4
4
68
69
3
.8. Kinetics of the metabolism of 20(OH)D3 and 20,23(OH)
2
D3 in
Fig. 7. Time course for the metabolism of 20,23-dihydroxyvitamin D3 in
phospholipid vesicles by CYP24A1. (A) Human CYP24A1 (0.05 M) and (B) rat
CYP24A1 (1 M) were incubated with 20,23(OH) D3 (at a molar ratio of 0.05 mol/
mol phospholipid) in a reconstituted system with adrenodoxin and adrenodoxin
reductase. The samples were analysed using HPLC Program B (see Section 2.3).
Product numbers refer to the peaks shown in Fig. 4.
phospholipid vesicles by CYP24A1
m
m
2
4
4
4
4
4
70
71
72
73
74
Previously we reported the kinetic parameters for the
metabolism of 1,25(OH) D3 and its C24-oxidation pathway
intermediates by human CYP24A1 [35]. In the current study, we
2
observed that 20(OH)D3 was metabolized with a kcat of 16.8 Æ 1.1
mol/min/mol CYP24A1 and
a
K
m
of 0.031 Æ0.005 mol/mol
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
expression of rat CYP24A1 than the human enzyme, enabling us to
use higher final concentration of P450, rat CYP24A1 was used to
generate more downstream metabolites from 20,23,24
475
476
477
478
479
480
phospholipid (Fig. 8). The 20,23(OH)
2
D3 substrate was metabo-
lized with similar kinetic values; kcat = 19.5 Æ 2.1 mol/min/mol
CYP24A1 and K
= 0.045 Æ 0.014 mol/mol phospholipid (Fig. 8). On
the basis of catalytic efficiency, 20(OH)D3 and 20,23(OH) D3 are
both relatively poor substrates for human CYP24A1 being metab-
olized with kcat/K
m
(
3 3
OH) D3 and 20,23,25(OH) D3 for mass spectral analysis. The
2
human CYP24A1 enzyme was used at a final concentration that
was 20-fold lower than for rat CYP24A1, but this still permitted
some of the earlier metabolites to be observed (Fig. 4F and H).
À1
m
values of 560 and 433 min (mol substrate/mol
3
When 20,23,25(OH) D3 incorporated into phospholipid
vesicles was incubated with rat CYP24A1, two major products
were observed (Fig. 4E). These were labeled as Products 2 and 5A,
based on alignment of their HPLC retention times with products
from the reaction of CYP24A1 on 20,23(OH)
After the 30 min incubation with 1 M rat CYP24A1, 32% of the
substrate remained and Product 5A accounted for 75% of the total
products formed. Incubation of 20,23,25(OH) D3 with human
2
D3 (Fig. 4A and C).
m
3
CYP24A1 yielded only Product 2 (Fig. 4F), suggesting that it is a
primary product that may give rise to Product 5A. As described
earlier, it was tentatively identified as a dehydro-trihydroxyvita-
min D3 species based on its mass spectrum. Product 5A was
tentatively identified as 20-hydroxy-23-carboxy-24,25,26,27-tet-
ranorvitamin D3 by high resolution mass spectrometry with an
exact mass of 374.2457. The mass spectrum gave ion fragments
with m/z = 357.2429, 339.2329 and 321.2215, which correspond to
+
+
+
[
(
M + H–H
Table 1, Fig. S3). This identification provides strong evidence that
D3 between
2 2 2
O] , [M + H–2H O] and [M + H–3H O] , respectively
Fig. 8. 20-Hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 are metabolized
with similar K and kcat values by human CYP24A1. Secosteroids at varying ratios to
phospholipid were incorporated into phospholipid vesicles and incubated with
human CYP24A1 (0.02 and 0.05 for 20(OH)D3 and 20,23(OH) D3,
respectively) for 2 min in a reconstituted system containing adrenodoxin and
adrenodoxin reductase. The products were analysed by HPLC using Program A for 20
m
CYP24A1 can cleave the side chain of 20,23(OH)
2
C23 and C24 subsequent to other oxidation steps, and then oxidize
the product to the C23 carboxylic acid, similar to the final steps in
mM
mM
2
the C24-oxidation pathway of 1,25(OH)
2
D3 metabolism [1,35].
(OH)D3 and HPLC Program B for 20,23(OH)
were fitted by non-linear least squares analysis by Kaleidagraph 4.0. The r values
were 0.998 and 0.988 for the curve fit for 20(OH)D3 and 20,23(OH) D3, respectively.
20,23(OH) D3; * 20(OH)D3.
2
D3 (see Section 2.3). Hyperbolic curves
2
0,23,24(OH) D3 was observed to be almost completely
3
metabolized by rat CYP24A1 and gave one major and several
minor products (Fig. 4G). The main product observed was Product
2
&
2
Please cite this article in press as: E.W. Tieu, et al., Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human
CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010

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11
4
4
81
82
À1
518
519
phospholipid) , respectively, which are markedly lower than for
therefore evident that CYP24A1 prefers hydroxylating the secos-
teroid at the more terminal carbons of the vitamin D side chain
when a 23-hydroxyl group is present. This is seen in the C23-
oxidation pathway of 25(OH)D3 metabolism where after initial
C23 hydroxylation, the next hydroxylation is at C26, with
C25 already having a hydroxyl group present [1,3].
2
the metabolism of 25(OH)D3 or 1,25(OH) D3 [35] (see Section 4).
5
20
521
22
4
83
4. Discussion
5
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
The novel vitamin
D metabolic pathway catalyzed by
CYP11A1 produces a number of secosteroids with hydroxyl groups
along the C20-27 side chain [9,10,15]. The most extensively
characterized secosteroids produced from this pathway with
The present study shows that human CYP24A1 can act on 20
(OH)D3 and 20,23(OH)
thus comparable catalytic efficiencies (kcat/K
kinetics of 20(OH)D3 metabolism by rat CYP24A1 to human
CYP24A1 in the phospholipid vesicle system, we found that the K
2
D3 with similar K
m
and kcat values, and
m
). In comparing the
respect to biological activity are 20(OH)D3 and 20,23(OH)
2
D3
[16,22,23,25–28,32–34,46,47]. In the present study, we show that
m
these two biologically active secosteroids can be metabolized by
both rat and human CYP24A1. The rat and human enzymes
produce the same major products from 20(OH)D3 and both
isoforms can hydroxylate at the two alternate C24 positions of this
values are comparable (0.028–0.031 mol substrate/mol phospho-
lipid) but human CYP24A1 had a 1.6-fold higher kcat than rat
CYP24A1. Previously we reported the kinetic parameters for the
metabolism of 25(OH)D3 and 1,25(OH) D3 by human
2
CYP24A1 using the same phospholipid-vesicle reconstituted
system employed in the current study, thus permitting a direct
chiral center to produce the two enantiomers of 20,24(OH)
Interestingly, human CYP24A1 prefers to initially hydroxylate 20
OH)D3 at C25 whereas rat CYP24A1 favors initial hydroxylation at
2
D3.
(
comparison of values. 1,25(OH)
2
D3 is metabolized by human
C24. This is not surprising due to the known differences between
the rat and human enzymes where rat CYP24A1 catalyses the C24-
oxidation pathway, while human CYP24A1 can catabolize vitamin
D through the C23- and C24-oxidation pathways. Metabolism of 20
CYP24A1 with kcat and K values that are 1.6-fold higher and 7-fold
m
lower than that for 20(OH)D3, respectively, with an overall 11-fold
higher catalytic efficiency. The relative efficiency for human
CYP24A1 is even higher for 25(OH)D3 where kcat/K
m
is 34-fold
(
1
(
[
2
OH)D3 by CYP24A1 is clearly different from that of 25(OH)D3 and
,25(OH) D3, as no subsequent oxidations were observed with 20
OH)D3, with all products being dihydroxyvitamins D3 species
33]. The presence of the 20-hydroxyl group prevents
3-hydroxylation as no 20,23(OH) D3 was seen among the
higher than for 20(OH)D3. In contrast, 24,25(OH) D3, the first
2
2
reaction intermediate of the C24-oxidation pathway of 25(OH)
D3 metabolism, is oxidized by human CYP24A1 with a catalytic
efficiency only 3-fold higher than for 20(OH)D3, and actually
2
displays a 3-fold higher K
C23 hydroxyl group to 20(OH)D3 to form 20,23(OH)
m
value [35]. Overall, the introduction of a
D3 does not
products [33]. Rather, the presence of the 20-hydroxyl group
shifts the position of the side chain in the active site such that 20,25
2
alter the kinetic parameters for the initial rates compared to those
seen with only the C20 hydroxyl group present. Having the
hydroxyl group at C20 rather than at C25 decreases the K ,
m
suggesting it weakens binding to the active site of the enzyme, and
reduces the overall catalytic efficiency dramatically.
(
(
OH)
2
D3 is the one of the major products of CYP24A1 action on 20
OH)D3. The sites of hydroxylation in the minor products remain to
be established.
Like 20(OH)D3, 20,23(OH)
2
D3 is also hydroxylated at C24 and
D3 and 20,23,25
D3 as initial products for both the rat and human enzymes.
The introduction of a hydroxyl group at C23 of 20(OH)D3 (as in
0,23(OH) D3), shifts the favored site of hydroxylation by the rat
enzyme from C24 to C25, the site preferred by the human enzyme
with both 20(OH)D3 and 20,23(OH) D3 as substrates. It is
C25 by CYP24A1, producing 20,23,24(OH)
OH)
3
Even though the initial rates of metabolism of 20(OH)D3 and
(
3
20,23(OH)
hydroxyl group to 20(OH)D3 by CYP11A1, producing 20,23
(OH) D3, dramatically alters the metabolic pathway catalyzed
2
D3 by CYP24A1 are similar, the addition of the 23-
2
2
2
by this enzyme. Only a single hydroxyl group is added to various
positions of the side chain of 20(OH)D3, and no secondary
2
Fig. 9. Pathways illustrating the multiple reactions carried out on 20,23-dihydroxyvitamin D3 by CYP24A1. The potential for Product 2 to be an intermediate for production of
Product 5A remains to be established (dashed arrow).
Please cite this article in press as: E.W. Tieu, et al., Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human
CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010

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5
5
5
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5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
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00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
623
624
625
626
627
628
629
630
631
632
633
634
metabolites are observed. In contrast, following hydroxylation of
0,23(OH) D3 to 20,23,24(OH) D3 and 20,23,25(OH) D3,
CYP24A1 can further oxidize these primary metabolites in a
complex series of reactions that appear to include side chain
cleavage between C23 and C24. Identified steps in the likely
pathway are illustrated in Fig. 9. CYP24A1 initially metabolizes
on studies of other side chain hydroxylated 20(OH)D3 products
such as 17,20,23(OH) D3 [21,26], it is likely that primary products
of CYP24A1 action on 20,23(OH) D3 will remain active, but later
products with the side chain cleaved are likely to be inactive, as is
observed for calcitroic acid [1,4].
In conclusion, this study shows that both human and rat
CYP24A1 hydroxylate 20(OH)D3 to produce only dihydroxyvita-
min D3 derivatives, which retain biological activity. 20,23
2
2
3
3
3
2
20,23(OH)
2
D3 to the primary products, 20,23,24(OH)
3
D3 and
20,23,25(OH)
3
D3, where the proportion of each primary product is
species dependent. Subsequent metabolism of 20,23,24
OH) D3 yields two different products which have three hydroxyl
groups and one keto-group on the side chain and maybe either 23-
oxo-20,24,X(OH) D3, 24-oxo-20,23,X(OH) D3 or X-oxo-20,23,24
OH) D3. Note that the tertiary hydroxyl groups at C20 or
(OH)
is analogous to the C24-oxidation pathway of 1,25
(OH) D3 metabolism, with evidence for side chain cleavage
2
D3 undergoes multiple oxidation steps in a pathway that
(
3
2
3
3
between C23 and C24.
(
3
635
C25 cannot be oxidized to a ketone. It is also possible that the
two dehydro-tetrahydroxy- products (Products 5B and 6) are
stereoisomers as this study shows that CYP24A1 can catalyze the
Acknowledgments
Q7 636
637
638
639
640
641
642
643
644
645
646
647
648
We would like to acknowledge The University of Western
Australia and the Ana Africh Postgraduate Scholarship in Medical
Research for their financial support. This work was partially
supported by NIH grants R21AR066505-01A1 and R01AR052190 to
AS. In addition there was financial support from NIH grants
1R21AR063242-01A1, 1S10OD010678-01, and RR-026377-01 to
WL. We thank Mr Min Xiao at the University of Tennessee Health
Science Center for help in preparing the samples for NMR
measurements. Low resolution mass spectral analysis performed
by the UWA Centre of Metabolomics was supported by infrastruc-
ture funding from the Western Australian State Government in
partnership with the Australian Federal Government, through the
National Collaborative Research Infrastructure Strategy (NCRIS).
formation of the two 20,24(OH)
High resolution mass spectrometry of Product 5B derived from
rat CYP24A1 action on 20,23,25(OH) D3, suggests that it is 20-
2
D3 enantiomers from 20(OH)D3.
3
hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D3. This is anal-
ogous to the final product of the C24-oxidation pathway of 1,25
(
2
OH)
4,25,26,27-tetranorvitamin D3) but with a 20-hydroxyl group
rather than a 1 -hydroxyl group. This indicates that CYP24A1 can
cleave the side chain of 20,23(OH) D3 and oxidize the product
likely to be 20-hydroxy-23-oxo-24,25,26,27-tetranorvitamin D3)
to the C23 carboxylic acid, as occurs in the metabolism of 1,25
OH) D3 by the C24-oxidation pathway [1,35]. The likely substrate
for the cleavage reaction is 24-oxo-20,23,X(OH) D3 which has the
2
D3 metabolism, calcitroic acid (1a-hydroxy-23-carboxy-
a
2
(
(
2
3
649
adjacent ketone and hydroxyl groups required for C23—C24 bond
cleavage, but the presence of this intermediate remains to be
established. It may also be possible for cleavage to occur between
Appendix A. Supplementary data
650
651
652
Supplementary data associated with this article can be found, in
the C23 and C24 in 23-oxo-20,24,X(OH)
identification of 20-hydroxy-23-carboxy-24,25,26,27-tetranorvi-
tamin D3 by the action rat CYP24A1 on 20,23,25(OH) D3 provides
3
D3. While the tentative
the
online
version,
at
http://dx.doi.org/10.1016/j.jsbmb.
2015.02.010.
3
653
evidence for C23-C24 bond cleavage, this intermediate was not
seen for human CYP24A1. However, this is likely to be due to the
low concentrations of human CYP24A1 used in these experiments
compared to rat CYP24A1, due to its low expression and the
difficulty in purifying substantial amounts of the active enzyme
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[
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Please cite this article in press as: E.W. Tieu, et al., Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human
CYP24A1, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.org/10.1016/j.jsbmb.2015.02.010