3-Epi-25-hydroxyvitamin D3 is a poor substrate for SULT2A1: Analysis of its 3-sulfate in cord plasma and recombinant human SULT2A1 incubate

Article abstract of DOI:10.1016/j.steroids.2020.108695

A variety of metabolites derived from 25-hydroxyvitamin D₃ [25(OH)D₃], including its 3-epimer [Epi-25(OH)D₃] and 3-O-sulfate [25(OH)D₃-3S], is found in human plasma/serum. We hypothesized that the 3-O-sulfate of Epi-25(OH)D₃ [Epi-25(OH)D₃-3S] might be present in plasma/serum. Clarifying this point could improve our understanding of the metabolism of vitamin D₃. In this study, we first carefully analyzed the cord plasma samples by derivatization-assisted liquid chromatography/electrospray ionization-tandem mass spectrometry and demonstrated the occurrence of Epi-25(OH)D₃-3S in the plasma. However, the concentration ratio of Epi-25(OH)D₃-3S to 25(OH)D₃-3S (sulfated form) was infinitely lower than the ratio of Epi-25(OH)D₃ to 25(OH)D₃ (unconjugated form). To determine what caused this result, we next performed an in vitro experiment of the 3-O-sulfation for 25(OH)D₃ and Epi-25(OH)D₃ using the recombinant human sulfotransferase (SULT) 2A1. This in vitro experiment revealed that Epi-25(OH)D₃ is a poor substrate for the 3-O-sulfation catalyzed by SULT2A1 as compared to 25(OH)D₃. This substrate specificity of SULT2A1 would be the main cause for the result obtained from the analysis of the cord plasma samples.

Full text of DOI:10.1016/j.steroids.2020.108695

Steroids162(2020)108695
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3-Epi-25-hydroxyvitamin D3 is a poor substrate for SULT2A1: Analysis of its
3-sulfate in cord plasma and recombinant human SULT2A1 incubate
⁎
Yusuke Yoshimuraa, Moeka Togashia, Shoujiro Ogawaa,b, Tatsuya Higashia,
a Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan
b Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Sanzo, Gakuen-cho, Fukuyama-shi, Hiroshima 729-0292, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
A variety of metabolites derived from 25-hydroxyvitamin D3 [25(OH)D3], including its 3-epimer [Epi-25(OH)D3]
and 3-O-sulfate [25(OH)D3-3S], is found in human plasma/serum. We hypothesized that the 3-O-sulfate of Epi-
25(OH)D3 [Epi-25(OH)D3-3S] might be present in plasma/serum. Clarifying this point could improve our un-
derstanding of the metabolism of vitamin D3. In this study, we first carefully analyzed the cord plasma samples
by derivatization-assisted liquid chromatography/electrospray ionization-tandem mass spectrometry and de-
monstrated the occurrence of Epi-25(OH)D3-3S in the plasma. However, the concentration ratio of Epi-25(OH)
D3-3S to 25(OH)D3-3S (sulfated form) was infinitely lower than the ratio of Epi-25(OH)D3 to 25(OH)D3 (un-
conjugated form). To determine what caused this result, we next performed an in vitro experiment of the 3-O-
sulfation for 25(OH)D3 and Epi-25(OH)D3 using the recombinant human sulfotransferase (SULT) 2A1. This in
vitro experiment revealed that Epi-25(OH)D3 is a poor substrate for the 3-O-sulfation catalyzed by SULT2A1 as
compared to 25(OH)D3. This substrate specificity of SULT2A1 would be the main cause for the result obtained
from the analysis of the cord plasma samples.
Sulfation
3-Epi-25-hydroxyvitamin D3
Cord plasma
SULT2A1
Substrate specificity
1. Introduction
25(OH)D3 into a metabolite that is excreted into the urine.
25(OH)D3 is also converted into its 3-epimer, 3-epi-25-hydro-
Vitamin D3 is metabolically activated to 1α,25-dihydroxyvitamin D3
[1,25(OH)2D3] by the sequential 25- and 1α-hydroxylation, which are
catalyzed by cytochrome P450 (CYP) 2R1 in the liver and CYP27B1 in
the kidney, respectively. 1,25(OH)2D3 plays several biological roles in
humans, such as the maintenance of calcium homeostasis and bone
formation. 25-Hydroxyvitamin D3 [25(OH)D3, Fig. 1a] occurs as the
major circulating form of vitamin D3 as well as the intermediate in this
metabolic activation of vitamin D3. The bio-conversion to
1α,25(OH)2D3 from 25(OH)D3 is strictly regulated by the serum cal-
cium level; 1α,25(OH)2D3 is reported to be in the plasma/serum at a
concentration of up to 150 pM, which is almost 1,000 times lower than
the normal concentration of 25(OH)D3 (40–100 nM) [1,2]. The excess
25(OH)D3 is partly converted to inactive and/or conjugated metabo-
lites. The major inactivation pathway of 25(OH)D is the CYP-dependent
side-chain oxidation, including the conversion into 24,25-dihydrox-
yvitamin D3 [24,25(OH)2D3]. The plasma/serum concentration of
24,25(OH)2D3 has been reported to be about one-tenth that of 25(OH)
D3 [1,2]. We recently identified 24,25(OH)2D3 24-glucuronide as the
major urinary vitamin D3 metabolites in humans [3]; this finding in-
dicates that the 24-hydroxylation is the first step in the conversion of
xyvitamin D3 [Epi-25(OH)D3, Fig. 1a], whose serum/plasma con-
centration has been reported to be an average of several percent that of
25(OH)D3 [4,5]. A recent study demonstrated that the C3-epimeriza-
tion of 25(OH)D3 is primarily catalyzed by 25(OH)D3 3-epimerase
present in liver microsomes [6]. It has also been reported that the
concentration ratio of Epi-25(OH)D3 to 25(OH)D3 in the plasma/serum
is higher in infants than in adults [7,8]. The 3-epimerization is thought
to be one of the metabolic pathways to reduce the biological activity of
vitamin D metabolites [9].
25(OH)D3 3-sulfate [25(OH)D3-3S, Fig. 1a] is another major circu-
lating metabolite of vitamin D3, and its plasma level (mean, ca. 60 nM)
was found to be much higher than that of 25(OH)D3 (mean, ca 20 nM)
in newborns [10,11]. 25(OH)D3-3S has a high binding affinity for the
vitamin D binding protein [12], which would be the main cause for its
relatively high abundance in the circulation. Recently, human sulfo-
transferase (SULT) 2A1 in the liver was identified as the major enzyme
responsible for the 3-O-sulfation of 25(OH)D3 [12,13]. 25(OH)D3-3S
might be the storage form of vitamin D3 because 25(OH)D3 can be re-
generated from this sulfate by deconjugation.
Thus, a variety of metabolites derived from 25(OH)D3 is found in
⁎ Corresponding author.
E-mail address: higashi@rs.tus.ac.jp (T. Higashi).
https://doi.org/10.1016/j.steroids.2020.108695
Received 29 May 2020; Received in revised form 25 June 2020; Accepted 30 June 2020
Availableonline07July2020
0039-128X/©2020ElsevierInc.Allrightsreserved.

Y. Yoshimura, et al.
Steroids162(2020)108695
Fig. 1. (a) Chemical structures of 25(OH)D3, Epi-25(OH)D3 and their sulfates and (b) energetically favored A-ring conformation of 25(OH)D3 and Epi-25(OH)D3.
human plasma/serum, and it is thought that the metabolism of 25(OH)
D3 plays a primary role in regulating the biological activities of vitamin
D3 in humans. As just described, 25(OH)D3-3S is the most abundant
circulating metabolite of vitamin D3 in newborns [10,11]. The plasma/
serum concentration ratio of Epi-25(OH)D3 to 25(OH)D3 is higher in
newborns/infants than in adults [7,8]. Taken these two findings to-
gether, the question about the presence or absence of Epi-25(OH)D3 3-
sulfate [Epi-25(OH)D3-3S] in the plasma/serum of newborns/infants
arose. Clarifying this question could improve our understanding of the
metabolism of vitamin D3.
HPLC [column, J’sphere ODS-H80 (YMC, Kyoto, Japan) and mobile
phase, methanol-10 mM ammonium formate (5:2, v/v)]. The chemical
structure of Epi-25(OH)D3-3S was confirmed by negative ESI-MS (m/z
479.1 [M−H]–) and UV spectroscopy (λmax 265 nm and λmin 228 nm),
and its purity determined by HPLC was over 99.5%.
[26,26,26,27,27,27-2H6]-25(OH)D3 [d6-25(OH)D3] and [26,26,26,27,
27,27-2H6]-25(OH)D3-3S [d6-25(OH)D3-3S] were purchased from Iso-
Sciences (King of Prussia, PA, USA) and used as the internal standards
(ISs). Standard solutions of the vitamin D3 metabolites and the ISs were
prepared by dissolving their precisely-weighed quantities in ethanol
and serial dilutions with ethanol. Recombinant human SULT2A1 and 3′-
phosphoadenosine 5′-phosphosulfate (PAPS) were purchased from R &
D systems (Minneapolis, MN, USA). Dithiothreitol (DTT) was from
Nacalai Tesque, Inc. (Kyoto). A HEPES buffer (50 mM, pH 7.5) was used
for the in vitro sulfation. DAPTAD was synthesized in our laboratories
[14]. An Oasis® HLB cartridge (30 mg adsorbent; Waters, Milford, MA,
USA) was used after successive washing with ethyl acetate (1 mL),
methanol (1 mL) and water (1 mL). The organic solvents and additives
used for the mobile phases were of LC/MS grade. All other reagents and
solvents were of analytical grade.
Based on this background information, we conducted two experi-
ments in this study. First, the presence or absence of Epi-25(OH)D3-3S
in umbilical cord plasma was examined by liquid chromatography/
electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). To
enhance the assay specificity, the derivatization using 4-(4-dimethyla-
minophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) (Fig. 2) [14] was
incorporated into the LC/ESI-MS/MS. Second, the in vitro sulfation ef-
ficiencies toward Epi-25(OH)D3 and 25(OH)D3 by the recombinant
human SULT2A1 were compared. The substrate specificity of the
human SULT2A1 was evaluated based on the amounts of the sulfate
formed in vitro.
2.2. LC/ESI-MS/MS
2. Experimental
A Waters Quattro Premier XE triple quadrupole-mass spectrometer
connected to an LC-e2695 chromatograph was used for the LC/ESI-MS/
MS analysis. The ESI-MS/MS parameters are described in the
Supplementary Data.
2.1. Materials and chemicals
25(OH)D3, Epi-25(OH)D3, 25(OH)D3-3S and 25-hydroxy-7-dehy-
drocholesterol 3-sulfate [25(OH)-7DHC-3S] were the same as those
used in previous studies [10,11]. 25-Hydroxyvitamin D3 25-sulfate
[25(OH)D3-25S] was obtained as a byproduct during the synthesis of
25(OH)D3-3S [10]. Epi-25(OH)D3-3S was synthesized from Epi-25(OH)
D3 in our laboratories according to a known method [15]. Briefly, Epi-
25(OH)D3 (230 μg, 0.5 μmol) was reacted with the sulfur trioxide-tri-
methylamine complex (1.0 mg, 7.2 μmol) in pyridine (100 μL) at room
temperature for 90 min, then the reaction mixture was purified by si-
lica-gel column chromatography [packing material, Wakogel® 60 N
(63–200 μm; FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan) and eluent, chroloform-methanol (4:1, v/v)] and reversed-phase
2.3. Cord plasma samples
In this study, newborn plasma was not available; as an alternative
sample, umbilical cord plasma was used because there are good cor-
relations in the concentrations between the newborn plasma and cord
plasma for both 25(OH)D3 and 25(OH)D3-3S [11]. The cord blood
samples were obtained during delivery at the Shizuoka Saiseikai Gen-
eral Hospital (Shizuoka, Japan) and initially used for the primary
clinical tests, then the remaining amounts of the samples were used for
this study. The plasma samples were prepared by centrifugation after
2

Y. Yoshimura, et al.
Steroids162(2020)108695
Fig. 2. Derivatization reaction scheme of Epi-25(OH)D3-3S with DAPTAD.
blood coagulation, then stored at –30 °C until used. The experimental
procedure was approved by the Ethics Committee of Tokyo University
of Science (No. 14001). Written informed consent forms were obtained
from all the subjects.
the derivatized d6-25(OH)D3.
For the methanol fraction, the solvent was evaporated, then the
residue was derivatized with DAPTAD in the same way as the ethyl
acetate fraction. The derivatized sample was dissolved in the mobile
phase C (60 μL), 15 μL of which was subjected to the LC/ESI-MS/MS
operating in the positive-ion mode. An Ascentis Express C18 column
(2.7 μm, 100 × 2.1 mm i.d., Merck Ltd., Tokyo, Japan) was used at the
flow rate of 0.2 mL/min and 40 °C. Methanol-10 mM ammonium for-
mate (3:2, v/v) containing 0.05% (v/v) formic acid was used as the
mobile phase C (isocratic elution). The SRM transitions were m/z
699.6 → 421.2 for the derivatized 25(OH)D3-3S and Epi-25(OH)D3-3S,
and m/z 705.6 → 421.2 for the derivatized d6-25(OH)D3-3S.
2.4. Analysis of cord plasma sample
A cord plasma sample (50 μL) was added to acetonitrile (100 μL)
containing d6-25(OH)D3 (1.0 ng) and d6-25(OH)D3-3S (1.0 ng), vortex-
mixed for 30 s, then centrifuged at 1,000 × g for 10 min. The super-
natant diluted with water (600 μL) was applied to the Oasis® HLB
cartridge. After washing with water (1 mL) and methanol–water (1:1,
v/v, 1 mL), the unconjugated vitamin D3 metabolites including 25(OH)
D3 and Epi-25(OH)D3 were eluted with ethyl acetate (1.5 mL). The
sulfated vitamin D3 metabolites including 25(OH)D3-3S and Epi-25(OH)
D3-3S were then eluted with methanol (1 mL).
2.5. Incubation procedure for in vitro sulfation
The in vitro sulfation procedure was based on the study by Kurogi
et al. [13]. The mixture of the substrate [25(OH)D3 or Epi-25(OH)D3,
200 ng in ethanol-HEPES buffer (2:23, v/v, 5 μL)], recombinant human
SULT2A1 [1.15 μg in HEPES buffer (5 μL)], PAPS [280 pmol in HEPES
buffer (5 μL)] and DTT [20 nmol in HEPES buffer (5 μL)], with a final
volume of 20 μL, was incubated in air at 37 °C for 10 min. Negative
control samples without the substrate or enzyme were assayed in par-
allel. The reaction was initiated by adding PAPS and terminated by
adding ice-chilled acetonitrile (50 μL) containing d6-25(OH)D3-3S
(50 ng).
For the ethyl acetate fraction, the solvent was evaporated, then the
residue was dissolved in ethyl acetate (50 μL) containing DAPTAD
(20 μg). After the derivatization was performed as previously reported
[11], the solvent was evaporated, then the resulting sample was dis-
solved in the mobile phase A (60 μL). Fifteen microliters of this sample
was injected into the LC/ESI-MS/MS operating in the positive-ion
mode. A YMC-Pack Pro C18 RS column (3 μm, 150 × 2.0 mm i.d.,
YMC) was used at the flow rate of 0.2 mL/min and 40 °C. The mobile
phase A [methanol-10 mM ammonium formate (7:2, v/v) containing
0.05% (v/v) formic acid] and mobile phase B [acetonitrile-10 mM
ammonium formate (9:1, v/v) containing 0.05% (v/v) formic acid]
were used with the following gradient elution program; B = 0%
maintained (0–6 min), 100% linearly increased (6–11 min) and main-
tained (11–13 min), and 0% maintained (13–29 min). The selected
reaction monitoring (SRM) transitions were m/z 619.5 → 341.3 for the
derivatized 25(OH)D3 and Epi-25(OH)D3, and m/z 625.5 → 341.3 for
2.6. Analysis of incubation sample
An incubation sample terminated with ice-chilled acetonitrile was
subjected to centrifugation at 1,000 × g for 10 min. The supernatant
was diluted with water (300 μL) and the sample was applied to the
Oasis® HLB cartridge. After washing with water (2 mL) and
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Y. Yoshimura, et al.
Steroids162(2020)108695
methanol–water (1:1, v/v, 1 mL), the remaining substrate [25(OH)D3
or Epi-25(OH)D3] was washed away with ethyl acetate (1.5 mL). The
formed sulfated vitamin D3 metabolite was then eluted with methanol
(1 mL). After the solvent was evaporated, the residue was derivatized
with DAPTAD and analyzed by LC/ESI-MS/MS. The LC conditions and
SRM transitions were the same as those for the analysis of the sulfated
vitamin D3 metabolites in the cord plasma.
well as 25(OH)D3-3S in the assay samples as their DAPTAD derivatives.
As previously reported, the 6R- and 6S-isomers are formed during
the DAPTAD derivatization for the vitamin D3 metabolites (Fig. 2) [14].
Therefore, two peaks appear on the chromatogram for some vitamin D3
metabolites after the derivatization. The 6R- and 6S-isomers of 25(OH)
D3-3S-DAPTAD co-eluted as
a single peak [retention time (tR)
28.1 min]. On the other hand, the 6R/S-isomers of Epi-25(OH)D3-3S-
DAPTAD separately eluted under the stated LC conditions and their tRs
were 25.7 and 30.5 min. The early-eluted peak (tR 25.7 min) was used
to identify Epi-25(OH)D3-3S in the cord plasma because the late-eluted
peak (tR 30.5 min) was hard to detect when the relatively large peak of
25(OH)D3-3S-DAPTAD appeared. For identification of Epi-25(OH)D3-3S
formed by in vitro sulfation, both of the peaks were used as will be
described later.
2.7. Quantification of vitamin D3 metabolites
The quantification of the vitamin D3 metabolites was carried out by
the previously-developed methods [11] with some modifications. These
methods were based on the internal standard method, in which d6-
25(OH)D3 and d6-25(OH)D3-3S were used as the ISs for the un-
conjugated and sulfated metabolites, respectively. The calibration
ranges for the cord plasma analysis were 1.0–50 ng/mL for 25(OH)D3,
0.1–2.0 ng/mL for Epi-25(OH)D3, 5.0–100 ng/mL for 25(OH)D3-3S and
0.4–8.0 ng/mL for Epi-25(OH)D3-3S. Those for the analysis of the in-
cubation sample were 1.0–20 ng/incubation sample for 25(OH)D3-3S
and 0.4–8.0 ng/incubation sample for Epi-25(OH)D3-3S.
3.2. Analysis of cord plasma sample
The unconjugated and sulfated vitamin D3 metabolites were frac-
tionated using an Oasis® HLB cartridge. In the model experiment, the
mixture of 25(OH)D3, Epi-25(OH)D3, 25(OH)D3-3S and Epi-25(OH)D3-
3S (1.0 ng each) were loaded on the cartridge, then a two-step elution
was performed. The ethyl acetate fraction of the first elution step
3. Results and discussion
contained 93.0
25(OH)D3, 94.1
2.4% [mean
standard deviation (SD), n = 3] of
3.1. LC/ESI-MS/MS behavior of DAPTAD-derivatized Epi-25(OH)D3-3S
5.2% of Epi-25(OH)D3, and no sulfated metabolites.
On the other hand, 84.7
2.9% of 25(OH)D3-3S, 86.0
4.2% of Epi-
It has been demonstrated that the DAPTAD derivatization enhances
the detectability and specificity in the positive-ESI-MS/MS not only for
the unconjugated vitamin D3 metabolites [14,16,17] but also for the
sulfated [11,12,18] and glucuronidated metabolites [3,18]. The deri-
vatization also works well for LC resolution of the stereoisomers, such
as 25(OH)D3 and Epi-25(OH)D3 [14,16]. These advantages prompted us
to use this derivatization to identify Epi-25(OH)D3-3S in the cord
plasma (Fig. 2).
25(OH)D3-3S and negligibly small quantities (≤0.2%) of 25(OH)D3 and
Epi-25(OH)D3 eluted into the methanol fraction of the second elution
step. Thus, our procedure could almost completely fractionate the un-
conjugated and sulfated metabolites.
The cord plasma samples were subjected to deproteinization fol-
lowed by the above fractionation using an Oasis® HLB cartridge. The
ethyl acetate fraction was first analyzed by LC/ESI-MS/MS after the
derivatization with DAPTAD. As shown in Fig. 4b, three peaks were
observed in the SRM chromatogram (m/z 619.5 → 341.3) of the cord
plasma sample; these peaks were identified as the DAPTAD-derivatized
25(OH)D3 (6R-isomer, tR 9.7 min), Epi-25(OH)D3 (tR 10.5 min) and
25(OH)D3 (6S-isomer, tR 11.3 min) by comparison to the authentic
standards (Fig. 4a) [14]. As is obvious from this chromatogram, Epi-
25(OH)D3 was definitely present together with 25(OH)D3 in the cord
plasma. The plasma concentrations of these metabolites were de-
termined by the internal standard method using d6-25(OH)D3 as the IS
Our first effort was directed toward an understanding of the mass
spectrometric and chromatographic behavior of the DAPTAD-deriva-
tized Epi-25(OH)D3-3S. Epi-25(OH)D3-3S-DAPTAD produced a proto-
nated molecule ([M + H]+) at m/z 699.6 in the positive ESI-MS. When
this ion was collisionally activated, a characteristic product ion at m/z
421.2 was efficiently produced similar to that for the derivatized
25(OH)D3-3S [11] (Fig. 3). Based on this result, the SRM transition of
m/z 699.6 → 421.2 was used for the detection of Epi-25(OH)D3-3S as
Fig. 3. Product ion spectrum of DAPTAD-derivatized Epi-25(OH)D3-3S.
4

Y. Yoshimura, et al.
Steroids162(2020)108695
Fig. 4. SRM chromatograms of DAPTAD-derivatized 25(OH)D3 and Epi-25(OH)D3 with IS [d6-25(OH)D3]. (a) Authentic standards; the mixture of 25(OH)D3 (1.0 ng)
and Epi-25(OH)D3 (100 pg) was derivatized, then one-quarter of the sample was injected into the LC/ESI-MS/MS. (b) Cord plasma sample. The measured con-
centrations of 25(OH)D3 and Epi-25(OH)D3 were 8.0 and 0.9 ng/mL, respectively.
(Table 1). The concentration ratio of Epi-25(OH)D3 to 25(OH)D3 was
9.3 1.3% (mean SD, n = 5).
The methanol fraction containing the sulfated metabolites was then
D3-3S. Although 25(OH)D3-25S and 25(OH)-7DHC-3S have not been
yet identified in human body fluids, we could not initially exclude the
possibility that the peak at 25.7 min might be derived from these
compounds. In the ESI-MS of the derivatized 25(OH)D3-25S, only the
sulfuric acid-eliminated ion ([M + H–H2SO4]+, m/z 601.5) was de-
tected. When this ion was collisionally activated, an ion at m/z 341.1
was produced by the cleavage of the C6-7 bond of the vitamin D ske-
leton (Fig. S1a, Supplementary Data). Thus, the derivatized 25(OH)D3-
25S provided quite different ions from the derivatized Epi-25(OH)D3-3S
in the ESI-MS and -MS/MS. Although the derivatized 25(OH)-7DHC-3S
provided its protonated molecule at m/z 699.6, the product ion formed
in MS/MS was observed at m/z 163.1 but not at m/z 421.2 (Fig. S1b,
Supplementary Data). These results verified that the derivatied 25(OH)
D3-25S and 25(OH)-7DHC-3S were not detected by the SRM transition
used for the detection of Epi-25(OH)D3-3S (m/z 699.6 → 421.2). In
other words, this SRM transition was highly specific for the detection of
analyzed as the DAPTAD derivatives. 25(OH)D3-3S was detected at a
significantly higher concentration (24.1
9.4 ng/mL, mean
SD,
n = 5) than its unconjugated form [25(OH)D3, 6.4
1.8 ng/mL] from
all the samples, which was consistent with previous studies [11]. The
occurrence of Epi-25(OH)D3-3S in the cord plasma was carefully ex-
amined according to the LC/ESI-MS/MS behavior described in Section
3.1. As shown in the chromatograms of Fig. 5b, a small peak (tR
25.7 min) was observed before the peak of the derivatized 25(OH)D3-
3S. This tR was completely identical to that of the authentic standard of
Epi-25(OH)D3-3S-DAPTAD (Fig. 5a).
To more reliably identify this peak, we examined the LC/ESI-MS/
MS behavior of the DAPTAD derivatives of 25(OH)D3-25S and 25(OH)-
7DHC-3S, whose molecular weights are the same as that of Epi-25(OH)
Table 1
Concentrations of unconjugated and sulfated forms of 25(OH)D3 and Epi-25(OH)D3 in cord plasma.
Sample
25(OH)D3 (ng/mL, A) Epi-25(OH)D3 (ng/mL, B) Ratio (B/A × 100, %) 25(OH)D3-3S (ng/mL, C) Epi-25(OH)D3-3S (ng/mL, D) Ratio (D/C × 100, %)
Plasma 1
Plasma 2
Plasma 3
Plasma 4
Plasma 5
Mean
6.4
8.0
0.5
0.9
0.7
0.6
0.3
0.6
7.8
11.3
9.5
9.0
9.1
9.3
36.2
27.2
28.1
14.3
14.8
24.1
Tracea
0.6
–
2.2
1.4
–
7.4
0.4
6.7
Tracea
Tracea
≤ 0.6
3.3
–
SD 6.4
1.8
0.2
1.3
9.4
≤ 2.2
a
Below limit of quantification (0.4 ng/mL).
5

Y. Yoshimura, et al.
Steroids162(2020)108695
Fig. 5. SRM chromatograms of DAPTAD-derivatized 25(OH)D3-3S and Epi-25(OH)D3-3S with IS [d6-25(OH)D3-3S]. (a) Authentic standards; the mixture of 25(OH)
D3-3S (1.0 ng) and Epi-25(OH)D3-3S (200 pg) was derivatized, then one-quarter of the sample was injected into the LC/ESI-MS/MS. (b) Cord plasma sample; the
intensity of the peak derived from Epi-25(OH)D3-3S (25.0–27.0 min) was enlarged 20 times. The measured concentrations of 25(OH)D3-3S and Epi-25(OH)D3-3S
were 27.2 and 0.6 ng/mL, respectively.
Epi-25(OH)D3-3S as well as 25(OH)D3-3S. Furthermore, the derivatied
25(OH)D3-25S (tR 26.9 min) and 25(OH)-7DHC-3S (tR 21.0 min) were
chromatographically separated from the derivatized Epi-25(OH)D3-3S.
Based on all the results considered, the peak at 25.7 min observed in the
cord plasma sample was almost certainly derived from Epi-25(OH)D3-
3S.
unconjugated form [Epi-25(OH)D3/25(OH)D3, 9.3
1.3%]. Based on
these results, we hypothesized that Epi-25(OH)D3 would be the poor
substrate in the 3-O-sulfation catalyzed by SULT2A1 compared to
25(OH)D3.
3.3. In vitro sulfation using recombinant human SULT2A1
Over the last decade, it has become clear that 20-hydroxyvitamin D3
[20(OH)D3], which is formed from vitamin D3 by action of CYP11A1
[2,19,20], is an active metabolite different from 1,25(OH)2D3; 20(OH)
D3 exerts anti-proliferative, pro-differentiation and anti-inflammatory
effects on epidermal cells [19,20]. 20(OH)D3 is found in human serum
[2,19] and the isomer of 25(OH)D3 and Epi-25(OH)D3. Assuming that
the 3-sulfate of 20(OH)D3 is present, it is the isomer of 25(OH)D3-3S
and Epi-25(OH)D3-3S. Although separation of the isomers is an im-
portant component for positive identification of the metabolites,
20(OH)D3 and its 3-sulfate could not be examined in this study due to
unavailability of their standards. However, our derivatization-based
LC/ESI-MS/MS method had a high capability for discrimination of
stereoisomers and positional isomers, such as 25(OH)D3-3S, Epi-25(OH)
D3-3S and 25(OH)D3-25S. Considering all the factors together, the
metabolites detected in the cord plasma samples (Fig. 4b and 5b) were
arguably identified as 25(OH)D3, Epi-25(OH)D3, 25(OH)D3-3S and Epi-
25(OH)D3-3S.
To test our hypothesis, we performed an in vitro experiment using
the recombinant human SULT2A1. The incubation sample was pre-
treated, derivatized, then subjected to LC/ESI-MS/MS. The recovery
rates of 25(OH)D3-3S and Epi-25(OH)D3-3S from the incubation sample
were almost equal; 86.1
88.3
3.4% (mean
SD, n = 3) and
3.9%, respectively. When 25(OH)D3 was incubated as the
substrate, a clear peak was observed at the elution position of the de-
rivatized 25(OH)D3-3S (tR 28.1 min, Fig. 6a). On the other hand, two
very small peaks appeared for the incubation sample with Epi-25(OH)
D3 as the substrate and their tRs (25.7 and 30.5 min) were identical to
those of the authentic derivatized Epi-25(OH)D3-3S (Fig. 6b). No peaks
corresponding to 25(OH)D3-3S and Epi-25(OH)D3-3S were detected in
the negative control samples. The amounts (per incubation sample) of
the formed sulfates were determined and summarized in Table 2. It
should be noted that compared to 25(OH)D3, Epi-25(OH)D3 was con-
verted into its sulfate at a significantly lower level; the conversion rate
The concentration of Epi-25(OH)D3-3S in the cord plasma was much
lower than that of 25(OH)D3-3S (Table 1); the plasma Epi-25(OH)D3-3S
concentrations were below the limit of quantification (0.4 ng/mL) in 3
of the 5 samples. Consequently, their ratio [Epi-25(OH)D3-3S/25(OH)
of Epi-25(OH)D3-3S from Epi-25(OH)D3 was 0.3
0.1% (mean
SD,
n = 5), which was about one tenth that of 25(OH)D3-3S from 25(OH)D3
(2.8
0.9%). Thus, Epi-25(OH)D3 appeared to be a poor substrate for
the 3-O-sulfation by SULT2A1 than 25(OH)D3, which indicated that
D3-3S,
≤ 2.2%] was significantly lower than the ratio of the
SULT2A1 exhibited a significant substrate specificity between 25(OH)
6

Y. Yoshimura, et al.
Steroids162(2020)108695
Fig. 6. SRM chromatograms of DAPTAD-derivatized 25(OH)D3-3S and Epi-25(OH)D3-3S formed in vitro. (a) Incubation sample with 25(OH)D3 as the substrate. The
formed amount of 25(OH)D3-3S was 7.6 ng/sample. (b) Incubation sample with Epi-25(OH)D3 as the substrate. The formed amount of Epi-25(OH)D3-3S was 0.8 ng/
sample.
compared to 25(OH)D3, which would be the main cause that the plasma
concentration ratio of Epi-25(OH)D3-3S to 25(OH)D3-3S was extremely
lower than expectation based on the concentration ratio of Epi-25(OH)
D3 to 25(OH)D3.
Table 2
In vitro conversion rates of 25(OH)D3 and Epi-25(OH)D3 to their sulfates by
SULT2A1.
Substrate
Amount of formed sulfatea (ng/
Conversion rateb (%)
incubation sample, A)
4. Conclusion
25(OH)D3
6.7
0.8
2.3
0.2
2.8
0.3
0.9
0.1
Epi-25(OH)D3
In this study, we found that Epi-25(OH)D3-3S is present in the cord
plasma at the trace level by using the specific LC/ESI-MS/MS method
combined with the DAPTAD derivatization. Interestingly, the plasma
concentration ratio of Epi-25(OH)D3-3S to 25(OH)D3-3S was much
lower than the ratio of Epi-25(OH)D3 to 25(OH)D3. To determine what
led to this result, the in vitro experiment using the recombinant human
SULT2A1 was performed. This experiment revealed that Epi-25(OH)D3
is a poor substrate for the 3-O-sulfation catalyzed by SULT2A1 as
compared to 25(OH)D3. These findings obtained in this study would be
of significant help for a deeper understanding of the vitamin D3 meta-
bolism.
a
b
Mean
SD (n = 5).
Conversion rate (%) = A (mean)/200 × 400/480 × 100. 200 is the
amount (ng) of the substrate. 400 and 480 are the molecular weights of the
substrate and its sulfate, respectively.
D3 and Epi-25(OH)D3.
Two pathways to produce 25(OH)D3-3S have been proposed by
Kurogi et al. [13]; (1) the direct 3-O-sulfation of 25(OH)D3, and (2) the
C25-hydroxylation of vitamin D3 3-sulfate (D3-3S). Because of the sig-
nificant low circulating concentration of D3-3S [21,22], we inferred
that the former would be the main pathway to produce 25(OH)D3-3S.
SULT2A1 has been identified as the most responsible enzyme for the
25(OH)D3-3S formation among the several SULT isoforms [12,13]. This
study found that the sulfating activity of SULT2A1 was much lower
toward Epi-25(OH)D3 than 25(OH)D3. The energetically favored con-
formers calculated by Chem3D MM2 (PerkinElmer, Waltham, MA) are
shown in Fig. 1b; the C3-hydroxy group is at the equatorial and axial
positions for 25(OH)D3 and Epi-25(OH)D3, respectively. The C3-hy-
droxy group of Epi-25(OH)D3 consequently might be prevented from
being located near the active site of SULT2A1. Our in vitro experiment
revealed that Epi-25(OH)D3 was much less sulfated by SULT2A1
CRediT authorship contribution statement
Yusuke Yoshimura: Methodology, Validation, Formal analysis,
Investigation, Resources, Data curation, Writing
- original draft,
Visualization. Moeka Togashi: Methodology, Validation, Formal ana-
lysis, Investigation, Resources, Data curation, Visualization. Shoujiro
Ogawa: Methodology, Investigation, Data curation, Writing - original
draft, Writing - review & editing. Tatsuya Higashi: Conceptualization,
Methodology, Writing - original draft, Writing - review & editing,
Visualization, Supervision, Project administration.
7

Y. Yoshimura, et al.
Steroids162(2020)108695
Declaration of Competing Interest
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1371/journal.pone.0018124.
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K. Mitamura, Development and validation of a method for determination of plasma
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trometry, J. Chromatogr. B 969 (2014) 230–234.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
[11] T. Higashi, M. Yokota, A. Goto, K. Komatsu, T. Sugiura, S. Ogawa, M. Satoh,
F. Nomura, A method for simultaneous determination of 25-hydroxyvitamin D3 and
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proton-affinitive Cookson-type reagent, Mass Spectrom. (Tokyo) 5 (2016) S0051,
https://doi.org/10.5702/massspectrometry.S0051.
Acknowledgments
The authors thank Dr. Takahiro Sugiura (Toyohashi Municipal
Hospital, Toyohashi, Japan) for collection of the cord plasma samples.
[12] T. Wong, Z. Wang, B.D. Chapron, M. Suzuki, K.G. Claw, C. Gao, R.S. Foti, B. Prasad,
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Appendix A. Supplementary data
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compounds–identification and characterization of the responsible human cytosolic
sulfotransferases, FEBS Lett. 591 (2017) 2417–2425.
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