Metabolism of [6,7-3H, 35S] estradiol 17-sulfate in rats

Article abstract of DOI:10.1016/S0039-128X(03)00037-0

To confirm whether or not the sulfo group of estradiol 17-sulfate (ES) is removed during in vivo metabolism in rats, the doubly labeled conjugate [6,7-³H, ³⁵S] ES was injected into rats, and its biliary and urinary metabolites were d

Full text of DOI:10.1016/S0039-128X(03)00037-0

Steroids 68 (2003) 383–392
3
35
Metabolism of [6,7- H, S] estradiol 17-sulfate in rats
∗
Kaori Takanashi, Yoshimi Itoh, Kazuhiro Watanabe, Itsuo Yoshizawa
Hokkaido College of Pharmacy, 7-1 Katsuraoka-cho Otaru, Hokkaido 047-0264, Japan
Received 18 November 2002; received in revised form 3 March 2003; accepted 6 March 2003
Abstract
To confirm whether or not the sulfo group of estradiol 17-sulfate (ES) is removed during in vivo metabolism in rats, the doubly labeled
3
35
conjugate [6,7- H, S] ES was injected into rats, and its biliary and urinary metabolites were determined by reverse isotope dilution method
RIDM). In male rats, the major radioactivity was detected in biliary disulfate fraction, which was composed of mainly ES and its two
(
minor metabolites, 2-hydroxyestradiol 17-sulfate (2-OH-ES) and 2-methoxyestradiol 17-sulfate (2-MeO-ES). In female rats, in contrast,
the radioactivity was dispersed into three fractions:biliary monosulfate, biliary disulfate, and urinary monosulfate fractions (Frs.) In both
monosulfate Frs., 7␤-hydroxyestradiol 17-sulfate was detected as the major metabolite followed by 6␣-, 6␤-, and 15␤-hydroxyestradiol
1
7-sulfates. Like male rats, 2-OH-ES and 2-Meo-ES as the minor products were detected in biliary disulfate fraction. The isotope ratios of
ES and its metabolites in both sexes were essentially the same as that of the dose except that of 6␣-hydroxylated metabolite, which may
be derived from the loss of the tritium labeled at C6. These results confirm the occurrence of the direct metabolism of ES in rats.
©
2003 Elsevier Science Inc. All rights reserved.
Keywords: [6,7- H, 35S] estradiol 17-sulfate; Rat; Direct metabolism of estrogen sulfate; Sex difference
3
1
. Introduction
estradiol 17-sulfates (6␣-, 6␤-, 7␤-, and 15␤-OH-ES, res-
pectively, Fig. 1) were produced, accompanied by the above
two minor catechols.
Previously, we reported the presence of estradiol 17-
sulfate (ES, Fig. 1) in the urine of non-pregnant [1] and
pregnant [2] women, and in the blood of pregnant women
Judging from the experimental conditions, these products
are considered to be formed via the direct hydroxylation of
ES. However, a different metabolic pathway is predicted to
occur in the in vivo metabolism of ES. For example, such
a pathway as “deconjugation of ES at first, followed by
the hydroxylation of estradiol (E) produced, and finally a
reconjugation at C17 of monohydroxylated products” cannot
be excluded.
[
3]. This was the first discovery of ES in mammals. Later,
a similar estrogen sulfate, 2-hydroxyestradiol 17-sulfate
2-OH-ES, Fig. 1), was detected in the blood [4] and
urine [5] of pregnant women, and it was surmised that
-OH-ES during pregnancy is a feto-placental metabolite of
ES [6].
Both estrogen sulfates were found to be endogenous also
(
2
To confirm whether or not the in vivo Phase I reaction
of ES occurs without the removal of the conjugate group
in rats [7]. In addition, the hepatic microsomes of rats were
shown to possess the ability to convert ES into 2-OH-ES
3
35
at C17, the doubly labeled steroid [6,7- H, S] ES was
injected into rats, and the urinary and biliary products were
analyzed.
[8]. Thus, it became evident that the Phase I reaction of ES
occurs not only in human but also in rat.
Recently, we found a big sex difference in the metabolic
profiles of rat hepatic microsomal hydroxylation of ES
[9]. In male rats, 2-OH-ES was produced accompanied by
2. Experimental
two minor products 4-hydroxyestradiol 17-sulfate (4-OH-
ES, Fig. 1) and a metabolite the structure of which is still
unknown. In female rats, 6␣-, 6␤-, 7␤-, and 15␤-hydroxy-
2.1. Materials
Estradiol was purchased from Steraloids (Wilton, NH,
∗
USA), and their 17-sulfates were prepared in this labo-
ratory using the method described previously [8,10]. The
Corresponding author. Tel.: +81-134-62-1876; fax: +81-134-62-5161.
E-mail address: yosizawa@hokuyakudai.ac.jp (I. Yoshizawa).
0
039-128X/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0039-128X(03)00037-0

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K. Takanashi et al. / Steroids 68 (2003) 383–392
2.3. HPLC
HPLC was carried out using pumps, Model CCPE
(
(
Tosoh, Tokyo) equipped with Model UV-8011 UV detector
280 nm, Tosoh). Analytical (anal.) HPLC was carried out
with ODS-80Tm (250 mm × 4.6 mm, i.d., Tosoh) as the sta-
◦
tionary phase at 40 C using a mixture of 0.5% ammonium
phosphate buffer solution (pH 3.0) and methanol (60:40,
v/v) as the mobile phase at a flow rate of 1.0–1.1 ml/min.
Preparative (prep.) HPLC was performed with ODS-80Tm
(
4
300 mm × 7.8 mm, i.d., Tosoh) as the stationary phase at
◦
0 C using the above solution as the mobile phase at a
flow rate of 3.0 ml/min.
2
2
.4. Preparation of radioactive estrogen sulfates
3
.4.1. [6,7- H] ES
A pyridine solution (3 ml) of chlorosulfonic acid (0.3 g)
◦
was warmed for 30 min at 50 C, and pyridine (0.1 ml) con-
3
taining [6,7- H] E was added. The mixture was allowed to
stand at 50 C for 60 min. Removal of the solvent in vacuo
Fig. 1. Chemical structures of ES and its derivatives. Dashes in the table
mean hydrogen (H).
◦
gave a residue, that was neutralized by 0.1 M sodium bicar-
bonate, followed by passing through Sep-Pak C18 cartridges.
After washing with water, a steroid-containing fraction was
obtained by elution with methanol. The methanolic eluate
was concentrated in vacuo to give a residue (corresponding
to EdiS), and this was dissolved in 5 ml of acetate buffer solu-
tion (100 mM, pH 5.0) containing arylsulfatase (80 units/ml).
preparation of 2-methoxyestradiol-17-sulfate (2-MeO-ES,
Fig. 1) was carried out by a known method [11]. Mono-
hydroxylated ES derivatives, 4-OH-ES [12], and 6␣-, 6␤-,
7
␣-, and 7␤-OH-ES (Fig. 1) [13], were prepared in this
laboratory according to known methods. The new sulfate
compound, 15␤-OH-ES, was prepared from 15␤-OH-E
◦
The whole mixture was incubated at 37 C for 3 days. Af-
[
(
14] by its partial sulfonation [15]. Estradiol disulfate
EdiS) was obtained from E according to the method of
ter filtration, the mixture was eluted through a Sep-Pak C18
cartridge. The methanolic eluate was concentrated in vacuo,
followed by treatment with ion-exchange resin. The aqueous
solution of sulfate was again passed through Sep-Pak C18
cartridges. The steroidal fraction obtained by elution with
methanol was subjected to prep. HPLC. The radiochemical
purity of the desired tracer was shown to be 98.6% by the
reverse isotope dilution method (RIDM).
Kirdani [16]. All estrogen sulfates used were potassium
salts prepared by treatment with an ion-exchange resin.
3
The labeled E (6,7- H, 1724 MBq/mol) was obtained from
New England Nuclear (Boston, MA, USA) and its iso-
tope contents at C6 and C7 were 43% (6␣) and 57%
(
7␣), respectively. No further purification of this material
35
was carried out. The isotope reagent [ S] chlorosulfonic
acid (1568 MBq/mol) was purchased from Amersham
35
2
.4.2. [ S] ES
(UK). Sep-Pak C18 cartridges were obtained from Waters
35
A pyridine solution (1 ml) of [ S] chlorosulfonic acid was
Co. (Milford, MA, USA). Arylsulfatase (type III from
abalone entrails) and other general reagents were pur-
chased from Sigma Chemical Co. (St. Louis, MO, USA)
and Wako Pure Chemicals, Ltd. (Tokyo, Japan), respec-
tively. Dowex 50W-X8 (200–400 mesh) was purchased
from Dow Chemicals (Midland, MI, USA) and used by
◦
warmed for 30 min at 50 C. This mixture was treated by
the same way as that described above and the radiochemical
purity of the product was obtained as 99.1% by RIDM.
2.5. Animal experiments
packing it into a column (30 mm × 10 mm, i.d.) in the
Wistar rats (body weight: male, 290–320 g and female,
30–260 g) were anesthetized with diethyl ether and can-
+
K
form.
2
nulated via the jugular vein and the common bile duct. A
3
35
2
.2. Counting radioactivity
mixture of [6,7- H] ES (222 kBq) and [ S] ES (288 kBq)
dissolved in physiological saline (0.2 ml) was administered
3
35
Radioactivities were determined by a Packard Tricarb-2650
via the tail vein. The isotope ratio ( H/ S) was 0.77. Ani-
mals were kept in metabolic cages. At appropriate time inter-
vals after the injection, urine and bile samples were collected
over a 24-h period and their radioactivities were determined.
To all of the bile and urine samples were added ascorbic
or an Aloka LSC-1000 liquid scintillation spectrophotome-
ter. ASC-II (Amersham) was used as a scintillant. The
35
radioactivity of S obtained was corrected in every case by
using a standard 35S-sample.

K. Takanashi et al. / Steroids 68 (2003) 383–392
385
Plate 1. Flow chart showing the procedures for separation and product analysis of rat biliary and urinary metabolites of [6,7- H, 35S] ES. No radioactivity
3
∗
was observed in Frs. 1, 3, and 4 of biliary and urinary metabolites from male rats. RIDM: reverse isotope dilution method.
acid (ca. 1 ␮g/ml) as an antioxidant and catechols 2-OH-ES
and 4-OH-ES (1 ␮g each) as carriers to prevent the decom-
position of catechol metabolites [17]. The animal studies
were performed in adherence to the Guiding Principles
for the care and use of experimental animals in Hokkaido
College of Pharmacy (published in 1988, revised in 2001).
ammonia (100 ml). Ten-milliliter fractions were collected,
part of which were pipetted out for radioactivity counting.
Fraction numbers 11–18 and 21–30 were combined to give
monosulfate Fraction (Fr.) and disulfate Fr., respectively
(Fig. 2).
2.8. Analysis of monosulfate fraction (Plate 1)
2
.6. Extraction of free steroids
The monosulfate Fr. was then subjected to prep. HPLC to
analyze the components.
Bile or urine sample was extracted with the same volume
of diethyl ether three times. The combined organic layer
was then washed with distilled water (about one-fifth of the
extract), followed by drying on anhydrous sodium sulfate,
and finally evaporated under a stream of nitrogen to give a
residue, the radioactivity of which was determined.
The biliary monosulfate Fr. from female rats gave on chro-
matography two radioactive peaks that were separated by
prep. HPLC to give Frs. 1 and 2 (Fig. 3). On the other hand,
the biliary monosulfate Fr. from male rats gave only one
radioactive peak corresponding to Fr. 2.
2
.7. Separation of mono- and disulfate fractions (Plate 1)
Fraction 1 was divided into five equal aliquots, each of
which was diluted with five authentic sulfates (6␣-, 6␤-,
7␣-, 7␤-, or 15␤-OH-ES: ca. 20 mg for each aliquot). Each
solution was subjected once to prep. HPLC to collect the
radioactive eluates (1 ml each) having UV absorbance at
280 nm, which were passed through Sep-Pak C18 cartridges,
followed by treatment with ion-exchange resin, and finally
recrystallization in appropriate solvents [13,15] to determine
the constant specific radioactivity (RIDM) for each steroid.
As the radioactivity of Fr. 2 was higher than that of
Fr. 1, approximately one-tenth or one-twentieth of the ra-
dioactivity of Fr. 2 was used for dilution with authentic ES
(ca. 20 mg), followed by RIDM determination in aqueous
methanol [8].
Ethanol was added to the combined bile or urine sam-
ples to a final concentration of 80%, and the mixture was
centrifuged at 1500 × g for 15 min to separate the super-
natant from the precipitated fractions. The precipitate was
suspended in 80% ethanol and centrifuged again at 1500×g
for 15 min. This precipitate was discarded because of its low
radioactivity (less than 0.1% of the radioactivity of the su-
pernatant). The combined supernatant was concentrated in
vacuo, dried, and subjected to column (Al2O3) chromatogra-
phy (10 cm ×1.5 cm, i.d.) using the following eluents in the
order of ethanol (100 ml), a gradient of ethanol (150 ml) and
water (150 ml), water (100 ml), and finally 0.1% aqueous

3
86
K. Takanashi et al. / Steroids 68 (2003) 383–392
2.9. Analysis of disulfate fraction (Plate 1)
The disulfate Fr. in both male and female rats was dis-
solved in 100 mM acetate buffer solution (pH 5.0, 3 ml)
containing arylsulfatase. The mixture was incubated for
◦
6
0 min at 37 C, followed by passing through a Sep-Pak
C18 cartridge. The steroidal fraction obtained was subjected
to prep. HPLC, in which five radioactive peaks (Frs. 3–7 in
Fig. 4) appeared.
Because Frs. 5–7 were found to correspond to 2-OH-ES,
ES, and 2-MeO-ES, respectively, about 20 mg of authen-
tic compound to each of them was added. Each solution
was subjected to RIDM after prep. HPLC for chromato-
graphic removal of the impurities, followed by treatment
with Sep-Pak C18 cartridges and with ion-exchange resin.
No further experiments were conducted on Frs. 3 and 4
of male rats because of their low radioactivity.
3. Results
Fig. 2. Representative example of alumina chromatographic separation of
biliary metabolites of [6,7- H, S] ES in male rat. The eluents are as
follows: A, ethanol (100 ml); B, gradient of ethanol (150 ml) and water
3.1. Excretion profiles of radioactivity
3
35
After injection of the doubly labeled ES into rats, bile,
(150 ml); C, water (100 ml); and D, 0.1% aqueous ammonia (100 ml).
Ten-milliliter fractions were collected at the flow rate of 1.0 ml/min.
and urine samples were collected for 24 h. In Table 1, the
Fig. 3. Representative example of prep. HPLC separation of biliary monosulfate fraction from female rat. Three-milliliter fractions were collected at the
flow rate of 3.0 ml/min. Fr. 1, fraction numbers 5–14; Fr. 2, fraction numbers 24–31.

K. Takanashi et al. / Steroids 68 (2003) 383–392
387
Fig. 4. Representative example of preparative HPLC separation of arylsulfatase-hydrolyzed product of biliary disulfate fraction from female rat.
Three-milliliter fractions were collected at the flow rate of 3.0 ml/min. Fr. 3, fraction numbers 5–11; Fr. 4, fraction numbers 13–17; Fr. 5, fraction
numbers 18–21; Fr. 6, fraction numbers 24–31; and Fr. 7, fraction numbers 35–42.
cumulative radioactivities of 3H and 35S in bile and
urine, and their ratios ( H/ S) are summarized. Over
three-quarters of the isotopes administered were excreted in
In male rats, most of the radioactivity excreted was present
in the biliary fraction. In contrast, biliary radioactivity was
nearly of the same degree as the urinary one in female rats.
The isotope ratios of the biliary fraction in the first few
hours after injection were slightly higher than that of the
dose (0.77) in both male and female rats, but they gradually
became similar. The isotope ratio of the urinary fraction in
female rats was slightly lower than that of the dose.
The combined biliary or urinary samples were extracted
with diethyl ether, and less than 0.01% of the radioactiv-
ity of the dose was detected in the organic layer from any
samples, indicating no formation of free steroids from the
radioactive ES injected. Ethanol was then added to the
aqueous layer to obtain the supernatant containing steroidal
conjugate.
3
35
2
4 h in both male and female rats, and a big sex difference
in the excretion profile was recognized.
Table 1
Cumulative amounts of isotopes excreted into bile and urine after intra-
3
35
venous injection of [6,7- H, S] ES in rat, and their isotope ratios
Time (h)
Male
Female
3H
35S
3H/35
3H
35S
3H/35
S
S
Bile
0
1
2
3
4
6
9
1
–1
–2
–3
–4
–6
–9
–12
2–24
21.9
38.3
47.4
52.5
59.3
64.7
67.6
74.8
20.3
35.3
45.0
50.4
57.9
64.0
67.4
75.8
0.83
0.84
0.81
0.80
0.79
0.78
0.77
0.76
13.0
22.7
28.3
31.6
34.3
35.4
35.7
36.3
11.9
21.2
28.1
32.2
35.3
36.3
36.6
36.8
0.84
0.82
0.78
0.76
0.75
0.75
0.75
0.76
3.2. Separation of mono- and disulfate fractions
The supernatant fraction was subjected to column chro-
matography on alumina. A representative chromatogram of
Urine
3
35
the biliary metabolite of [6,7- H, S] ES in male rat is
shown in Fig. 2, where small and large radioactive peaks
representing two isotopes are observed. By comparison with
such authentic steroids as ES or EdiS by anal. HPLC, the
0
1
–12
2–24
0
1.11
0
1.01
–
0.85
34.0
37.6
34.9
39.8
0.75
0.73
Amounts of isotopes are expressed as percentage of the dose (mean value,
n = 3).

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K. Takanashi et al. / Steroids 68 (2003) 383–392
Table 2
radioactivity detected in the disulfate Fr. In female rats, in
contrast, about half of the radioactivity excreted was found
in bile, with about 70 and 30% being the mono- and disul-
fate Frs., respectively.
Radioactivity (% of the dose) in biliary and urinary sulfate fractions and
their isotope ratios
Fraction
Male
Female
3H
35S
3H/35
3H
35S
3H/35
Regarding urinary metabolites, no disulfate was detected
in both male and female rats. The radioactivity in the urinary
monosulfate Fr. was about 1 and 48%, respectively, com-
pared to the dose in male and female rats. No further experi-
ment was carried out on the urinary fraction from male rats,
because its radioactivity was too low. No free estrogens or
their glucuronides were detected.
S
S
Biliary Fr.
Monosulfate
Disulfate
68.7
7.5
61.2
69.3
7.1
62.2
0.76
0.81
0.76
34.8
24.5
10.3
32.1
23.2
8.9
0.84
0.81
0.89
Urinary Fr.
Monosulfate
Disulfate
0.82
0.82
–
0.81
0.81
–
0.78
0.78
–
31.7
31.7
–
38.0
38.0
–
0.64
0.64
–
Amounts of isotopes are expressed as percentage of the dose (mean value,
n = 3).
3.3. Components of mono- and disulfate fractions
small and large peaks were surmised to be the mono- and
disulfate conjugates, respectively. These mono- and disul-
fate fractions were, thus, hereafter called monosulfate Fr.
and disulfate Fr., respectively.
The distribution of the radioactivity in the mono- and
disulfate Frs. obtained by alumina chromatography is sum-
marized in Table 2, where the biliary metabolites are ex-
creted as mono- and disulfates in both male and female rats.
In male rats, almost all of the two radioactivities excreted
were found in the biliary fraction, with about 90% of the
The components of the mono- and disulfate Frs. obtained
by alumina chromatography were then analyzed as follows.
In the case of female rats, biliary and urinary monosulfate
Frs. gave on prep. HPLC two radioactive peaks (Frs. 1 and 2),
as shown in Fig. 3. In male rats, almost only one radioactive
peak corresponding to Fr. 2 appeared. The disulfate Frs. of
both male and female rats were treated with arylsulfatase to
convert them into their monosulfates, the chromatographic
profiles of which revealed five radioactive peaks (Frs. 3–7),
as shown in Fig. 4.
Table 3
Examples of specific radioactivities of the biliary metabolites of [6,7- H, 35S] ES in a female rat determined by reverse isotope dilution method and
3
yields (%) of the metabolites from ES
Metabolite
(A) Amount of
metabolites used
for RIDM
(B) Weight of
carrier added
(mg)
(C) Specific radioactivity (dpm/mg)
(B × C/A) Total radioactivity of
3
3
of recrystallization product ( H,
metabolites ( H, dpm) and yield
3
3
35
×10 ) and ratio ( H/ S)
(%) of metabolites from ES
Monosulfate Fr.
ES
×1/20
×1
20.41
21.36
22.62
22.10
20.53
21.01
1st
2.30 (0.74)
2.65 (0.78)
2.67 (0.78)
1.36 (0.51)
1.01 (0.44)
0.99 (0.43)
3.68 (0.79)
3.40 (0.77)
3.38 (0.78)
0.29 (0.96)
0.02 (0.65)
0.01 (0.21)
2.80 (0.75)
2.89 (0.76)
3.00 (0.76)
5.89 (0.77)
6.51 (0.76)
6.60 (0.77)
2
3
nd
rd
6
1.09 × 10 (8.2)
6␣-OH-ES
6␤-OH-ES
7␣-OH-ES
7␤-OH-ES
15␤-OH-ES
1st
2
3
nd
rd
4
2.11 × 10 (0.016)
×1/2
×1
1st
2
3
nd
rd
5
1.53 × 10 (0.12)
1st
2
3
nd
rd
–
×1/20
×1/2
1st
2
3
nd
rd
6
1.23 × 10 (9.3)
1st
2
3
nd
rd
5
2.77 × 10 (2.1)
Disulfate Fr.
ES
×1/5
×1
21.22
30.46
31.20
1st
5.29 (0.77)
5.10 (0.76)
5.11 (0.76)
3.62 (0.78)
3.43 (0.77)
3.45 (0.77)
0.92 (0.78)
0.90 (0.76)
0.91 (0.76)
2
3
nd
rd
5
5.42 × 10 (0.41)
2
2
-OH-ES
1st
2
3
nd
rd
5
1.05 × 10 (0.08)
-MeO-ES
×1
1st
2
3
nd
rd
4
2.84 × 10 (0.02)

K. Takanashi et al. / Steroids 68 (2003) 383–392
389
Table 4
Isotope contents (%) in biliary or urinary metabolites determined by reverse isotope dilution method
Male
Female
3H
3H
7.2
4.1
–
35S
6.9
4.3
–
3H/35
S
35S
3H/35
S
Biliary monosulfate Fr.
ES
0.80
0.73
–
26.1
7.6
8.6
25.4
7.9
9.0
1.2
0.28
1.3
7.2
0.79
0.74
0.74
0.64
0.44
0.77
0.79
7␤-OH-ES
6␤-OH-ES
6␣-OH-ES
15␤-OH-ES
–
–
–
2.9
–
–
–
2.4
–
–
–
0.93
1.0
0.16
1.3
Others
7.4
Biliary disulfate Fr.
ES
65.5
54.7
2.5
1.5
6.8
67.3
57.2
2.4
1.6
6.1
0.75
0.74
0.80
0.72
0.86
10.4
4.2
0.72
0.18
5.3
10.1
4.4
0.73
0.20
4.8
0.79
0.74
0.76
0.69
0.85
2
2
-OH-ES
-MeO-ES
Others
Urinary monosulfate Fr.
ES
0.82
–
–
–
–
–
0.82
0.81
–
–
–
–
–
0.81
0.78
–
–
–
–
–
0.78
32.4
8.2
13.6
0.36
0.13
3.0
34.0
8.6
13.5
0.34
0.24
2.9
8.4
0.73
0.73
0.78
0.82
0.42
0.80
0.65
7␤-OH-ES
6␤-OH-ES
6␣-OH-ES
15␤-OH-ES
Others
7.1
Amounts of isotopes are expressed as percentage of the dose (mean value, n = 3).
Fractions 5–7 were identified by RIDM as 2-OH-ES, ES,
and 2-MeO-ES, respectively, in both sexes, the representa-
tive results of which are shown in Table 3.
In male rats, therefore, EdiS derived from ES accounted
for nearly 90% of the total radioactivity excreted in bile.
two-thirds and one-third of the biliary metabolites were
observed in the mono- and disulfate Frs., respectively.
From the biliary monosulfate Fr., ES (corresponding to
7.6% of the dose), 7␤-OH-ES (8.6%), 15␤-OH-ES (1.3%),
6␤-OH-ES (1.0%), and 6␣-OH-ES (0.16%) were detected.
These metabolites except 6␣-OH-ES had almost the same
3
.4. Quantification of metabolites
3
35
isotope ratios ( H/ S) ranging from 0.64 to 0.77, which
means essentially no loss of the sulfo group during the
hydroxylation. The low isotope ratio (0.44) of 6␣-OH-ES
may be caused by loss of tritium at C6 during the hydroxy-
lation. From the biliary disulfate Fr., ES (4.2% of the dose),
2-OH-ES (0.72%), and 2-MeO-ES (0.18%) were obtained
and found to have almost unchanged isotope ratios. This
means no loss of the sulfo group during the metabolism.
From the urinary monosulfate Fr., ES (corresponding to
8.2% of the dose), 7␤-OH-ES (13.6%), 15␤-OH-ES (3.0%),
6␤-OH-ES (0.36%), and 6␣-OH-ES (0.13%) were detected.
The isotope ratios of the products or that of ES recovered
had essentially the same values as that of the dose except
6␣-OH-ES. The low isotope ratio observed in 6␣-OH-ES
is explained by the same reason as that described in biliary
metabolites.
3
35
Table 4 presents the isotope contents and ratios ( H/ S)
3
35
in biliary and urinary metabolites of [6,7- H, S] ES as
determined by RIDM.
In male rats, the main metabolic route of ES was shown
to be sulfation, and about 84% of the radioactivity ex-
creted was EdiS. The isotope ratio of ES derived from
EdiS was 0.74, and was almost the same as that of the
dose. Other metabolic changes of ES, although of a lesser
extent, are 2-hydroxylation giving 2-OH-ES, followed by
2
-O-methylation giving 2-MeO-ES. The isotope ratios of
both metabolites were almost the same as to that of the
dose, meaning no loss of 35S during the hydroxylation fol-
lowed by O-methylation at C2. Although some radioactivity
remained in the biliary monosulfate Fr., no further work
was attempted because of the lack of authentic specimens.
About 4% of ES injected was recovered from the mono-
sulfate Fr. with an isotope ratio 0.74, which was almost
the same as that of the dose. Of interest is that in contrast
to female rats, the radioactivity in the urinary monosulfate
Fr. in male rats is extremely low (only 1% of the dose),
and that led us to abandon further experiments on this
fraction.
In all metabolites in both sexes, no formation of 7␣-hyd-
roxy metabolite (7␣-OH-ES) was confirmed and 4-OH-ES
was not detected because the amount produced was too small
for detection.
4. Discussion
In female rats, the radioactivities of the biliary and uri-
nary fractions were nearly of the same degree, and about
In the metabolism of ES by rat hepatic microsomes, hy-
droxylation occurs in the A, B, and D rings while retaining

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K. Takanashi et al. / Steroids 68 (2003) 383–392
the conjugated groups [9]. In order to investigate whether or
not this Phase I reaction occurs in vivo as well, a method that
was frequently employed in metabolic research on steroid
sulfates more than 30 years ago was used, which involves the
use of two types of labeled radioisotopes. As a few examples
of the “direct metabolism”, the conversion of androstene-3␤,
occurred to a considerable extent in female rats. In male rats,
although the amounts of the metabolites formed were only
a few percent of the injected dose, 2-OH-ES and 2-MeO-ES
were detected from the product of the arylsulfatase treatment
of the biliary disulfate Fr., and quantified by RIDM. The
isotope ratios of these two metabolites were nearly equal to
that of the dose (0.77), indicating that both 2-hydroxylation
of ES and 2-O-methylation of the formed catechol proceed
while retaining the C17-conjugated group. Furthermore, al-
though trace amounts of 4-OH-ES were produced in the case
of in vitro metabolism by hepatic microsomes [9], 4-OH-ES
could not be detected in this study, probably because the
amount produced was too small for detection.
The excretion of 2-MeO-ES as disulfate indicates that its
precursor is 2-OH-ES. Thus, a portion of 2-OH-ES formed
from ES is 2-MeO-ES, after which it is converted to disul-
fate. This indicates that a portion of 2-OH-ES that escaped
selective 2-O-methylation [21] is subjected to sulfate con-
jugation in the A ring. Thus, it is surmised that sulfate
conjugation of 2-OH-ES takes place either at C2 or C3.
2-Hydroxylation (and its subsequent 2-O-methylation) was
also observed in female rats, and 2-OH-ES and 2-MeO-ES
having the same isotope ratios were obtained by arylsulfa-
tase treatment of the biliary disulfate Fr. These results in-
dicate that in rats, in addition to the metabolic pathway of
ES → 2-OH-ES → 2-MeO-ES, there is also a pathway
leading to their disulfates.
1
7␤-diol 3-sulfate into dehydroepiandrosterone 3-sulfate in
humans [18], estrone sulfate into estradiol 3-sulfate in hu-
man fetus [19], or estrone sulfate into estradiol disulfate via
estradiol 3-sulfate in chicken [20] are mentioned. In this
3
35
study, [6,7- H, S] ES was injected into rats and its biliary
and urinary metabolites were examined.
As shown in Fig. 2, only two peaks corresponding to the
monosulfate and disulfate metabolites appeared in both bile
3
35
and urine samples, and the isotope ratios ( H/ S) of the
two peaks were nearly the same as that of the injected dose
(0.77) (Table 2). That means, apart from the Phase I reaction,
deconjugation was not found to occur in the metabolism of
ES, while conversely, sulfate conjugation was found to occur.
Since it was found that the metabolites of ES can be
broadly divided into two types comprising the mono- and
disulfate Frs., component analyses were performed on each
of these fractions by RIDM. The monosulfate Fr. was sepa-
rated into Frs.1 and 2 by HPLC, and since Fr. 2 was deter-
mined to be composed mainly of ES, quantitative determi-
nation was carried out by RIDM. Although Fr.1 was found
to correspond to at least five types of authentic sulfates
(
6␣-, 6␤-, 7␣-, 7␤-, and 15␤-OH-ES) according to anal.
The major sex difference in the in vivo metabolism of ES
in rats is the induction of hydroxylation of the B and D rings
observed only in females. These were found in the biliary
monosulfate and disulfate Frs., in which their isotope ratios
were nearly equal to that of the dose with the exception of
6␣-OH-ES (Table 4). The reason why 6␣-OH-ES exhibits
a low isotope ratio (0.46) in female bile and urine samples
HPLC, since their separation was not clear-cut, Fr.1 was
unavoidably equally divided into five aliquots and RIDM
was carried out after adding the above five authentic sulfates
to each aliquot, followed by prep. HPLC to remove the im-
purities. Treatment of each aliquot by prep. HPLC at once
made it easier to obtain high specific radioactive steroid.
After initially treating the disulfate Fr. with arylsulfatase,
it was separated into five monosulfate Frs. equivalent to Frs.
3
is believed to be as follows. Since the H content in the
labeled site of the steroid portion of injected ES was 43%
3
–7 by prep. HPLC. Since Frs. 5–7 were determined to be
for C6␣ and 57% for C7␣, the resulting calculation yields
a loss of 43% H by the product due to 6-hydroxylation,
3
equivalent to 2-OH-ES, ES, and 2-MeO-ES, respectively,
they were subjected to RIDM. Frs. 3 and 4 were not an-
alyzed due to their low levels of radioactivity. The biliary
metabolites of a female rat are shown in Table 3 as an exam-
ple of the measurement results obtained by RIDM for each
component of the mono- and disulfate Frs. In Fr. 1, since
the radioactivity of 7␣-OH-ES decreased rapidly with re-
peated recrystallization and did not exhibit constant values,
its formation could probably be ruled out. It was clear that
the other four types of monohydroxylated ES were formed.
provided that the isotope effects at C6 or C7 and so forth are
ignored. Thus, the isotope ratio of 6␣-OH-ES is theoretically
determined to be 0.44, and this can be considered to be equal
to the experimental value. On the other hand, the isotope
ratio (0.76) of the formed 7␤-OH-ES indicates that loss of
3
7␣- H does not occur due to 7␤-hydroxylation. Ultimately,
the Phase I reaction of ES can be said to consist entirely of
a “direct metabolism.”
A comparison of the results of this study with those
obtained in the in vivo metabolism of E in rats by Maggs
et al. [22] demonstrates that the presence or absence of
a conjugated group at C17 has a considerable effect on
estrogen metabolism. According to their findings, the ini-
tial metabolism of E involves the conversion into estrone
by oxidation at C17, and considerable sex differences
were observed in its biliary metabolites. In contrast to
the metabolism of estrone in female rats, which consists
mainly of 2-hydroxylation and 2-O-methylation of the
3
35
Their H/ S ratios were constant and almost the same as that
of the dose value (0.77). Similarly, the measurement of Frs.
5
2
–7 by RIDM revealed the formation of 2-OH-ES, ES and
-MeO-ES, respectively. Quantitative determination of ES
or its metabolites was also performed by RIDM in the same
manner on other rats, and the results are shown in Table 4.
The Phase I reaction of ES was proved to have consider-
able sex differences. Although C2-hydroxylation occurred
only slightly in male rats, hydroxylation of the B and D rings

K. Takanashi et al. / Steroids 68 (2003) 383–392
391
Plate 2. Potential metabolic pathway of estradiol 17-sulfate in rat. (
) Not observed in male rat.
resulting catechol form, the process is more complex in
male rats. In male rats, there are two parallel compositive
pathways of estrone hydroxylation, with one consisting
of the 15␣-hydroxylation of estrone and its subsequent
[2] Takanashi K, Yoshizawa I. Determination of urinary estradiol 17-
sulfate during pregnancy by a radioimmunoassay: urinary levels
throughout the menstrual cycle. Jap J Clin Chem 1992;21:26–9.
[
3] Honjo H, Tanaka K, Yasuda J, Ohno Y, Kitawaki J, Naito K, et al.
Serum estradiol 17-sulphate and lipid peroxides in late pregnancy.
Acta Endocr 1992;126:303–7.
2
-hydroxylation and 2-O-methylation, and the other con-
sisting of 16␣-hydroxylation of estrone and its subsequent
-hydroxylation and 2-O-methylation. The metabolic pro-
[4] Kashiwagi T, Yasuda J, Yamamoto T, Honjo H, Takanashi K,
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2
file in female rats is characterized by its extreme simplicity
in comparison with that in male rats. The hydroxylation
site of steroid sulfate by P450, being affected by the sulfate
ester group, was previously studied in detail by Gustafsson
and Ingelman-Sundberg [23,24]. The results obtained in
this study can be considered to belong to the same category
as this type of hydroxylation regulatory mechanism.
[
5] Takanashi K, Honma T, Kashiwagi T, Honjo H, Yoshizawa I.
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Conspicuous sex differences were observed even at the
point of conjugate formation in the metabolism of ES
(
Table 4). In the case of male rats, about 80% of the total
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1
dose was not involved in the Phase I reaction and the major-
ity was excreted as disulfate. In contrast, the percentage of
the total dose that was not involved in the Phase I reaction
in female rats was approximately one-third (approximately
microsomes. J Pharm Dyn 1982;5:340–7.
[
9] Itoh Y, Matsuda N, Matsuura K, Watanabe K, Takanashi K, Itoh
S, et al. On the structures of hydroxylated metabolites of estradiol
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6%) of that in male rats. Thus, although the Phase I reac-
[10] Watanabe K, Yoshizawa I. Induction of microsomal 2-hydroxylation
of estradiol 17-sulfate by phenobarbital in male rat. J Pharm Dyn
tion product of ES is required in female rats, it may not be
required in male rats. Although the physiological activity of
estrogen is typically lost following conjugation, it is known
that there is also a case in which the metabolite may have
new action [5].
On the basis of the results of this study that does not
involve the enterohepatic circulation, the entire aspect of
the metabolism of ES in rats can be represented by the
pathway shown in Plate 2. In contrast to the metabolism
in female rats consisting mainly of hydroxylation at C6␣,
C6␤, C7␤, and C15␤, with slight hydroxylation at C2
1983;6:438–40.
[
11] Watanabe K, Yoshizawa I. Preparation and high-performance liquid
chromatographic determination of guaiacol estrogen 17␤-conjugates:
the enzymatic O-methylation products of 2-hydroxyestradiol
17␤-conjugates (clinical analysis on steroids. XXII). Chem Pharm
Bull 1982;30:3231–8.
[
12] Watanabe K, Kimura R, Yoshizawa I. Clinical analysis on steroids.
XXIX. Evidence for 4-hydroxylation of estradiol 17-sulfate by rat
liver microsomes. Chem Pharm Bull 1984;32:2745–51.
[13] Itoh Y, Matsuda N, Harada K, Takanashi K, Watanabe K, Takagi H,
et al. Synthesis of 6- and 7-hydroxyestradiol 17-sulfates: the potential
metabolites of estradiol 17-sulfate by female rat liver microsomes.
Steroids 1999;64:363–70.
(and its subsequent O-methylation) along with their sul-
[14] Cantrall EW, Littell R, Bernstein S. The synthesis of C-15␤-
fate conjugation, the metabolism in male rats consists of
slight 2-hydroxylation and methylation, and mainly of the
formation of EdiS by sulfate conjugation of unchanged ES.
substituted estra-1,3,5(10)-trienes. II. J Org Chem 1964;29:214–7.
[15] Itoh Y, Itoh S, Takanashi K, Watanabe K, Yoshizawa I. In preparation.
[16] Kirdani RY. The synthesis and characterization of estradiol sulfates.
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[
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