Clinical Chemistry 46: 373-378, 2000;
(Clinical Chemistry. 2000;46:373-378.)
© 2000 American Association for Clinical Chemistry, Inc.
Detection and Measurement of Urinary 2-Hydroxyestradiol 17-Sulfate, a Potential Placental Antioxidant during Pregnancy
Kaori Takanashi1,
Takashi Honma1,
Tomohiro Kashiwagi2,
Hideo Honjo2 and
Itsuo Yoshizawa1,a
1
Hokkaido College of Pharmacy, 7-1, Katsuraoka-cho, Otaru 047-0264, Japan.
2
Department of Obstetrics and Gynecology, Kyoto
Prefectural University of Medicine, Kawaramachi, Hirokoji, Kamigyo-ku,
Kyoto 602-0000, Japan.
a Author for correspondence. Fax 81-134-62-5161; e-mail yosizawa{at}hokuyakudai.ac.jp
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Abstract
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Background: Preeclampsia is associated with a quantitative
imbalance between lipid peroxide and an antioxidant coproduced in the
placenta. To investigate our hypothesis that 2-hydroxyestradiol
17-sulfate (2-OH-ES) is the placental antioxidant during pregnancy, we
developed an assay for 2-OH-ES in urine and studied samples from women
with and without preeclampsia.
Methods: The detection and measurement of 2-OH-ES in the urine of
pregnant women were performed by RIA using highly specific
antiserum to 2-OH-ES. To confirm the reliability of the RIA method, the
same samples were analyzed by HPLC using an electrochemical detector.
Results: Urinary 2-OH-ES values obtained by RIA showed a close
relationship to those obtained by HPLC (y =
1.1x - 0.01; r = 0.96). The
urinary 2-OH-ES concentrations during the first, second, and third
trimesters were 2.0 ± 0.6 (mean ± SE, n = 13),
5.3 ± 1.3 (n = 21), and 15.3 ± 2.0 µg/mg creatinine
(n = 54), respectively, and <0.15 µg/mg creatinine (n =
10) at 2–24 h after delivery. The concentrations in preeclamptic women
during the third trimester were significantly lower, 3.9 ± 1.9
µg/mg creatinine (mean ± SE, n = 12).
Conclusions: RIA can be used to measure urinary 2-OH-ES during
pregnancy. The increase in urinary 2-OH-ES during gestation, its
decrease after delivery, and the lower values in preeclampsia are
consistent with a role of 2-OH-ES as a placental
antioxidant.
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Introduction
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Preeclampsia is a serious disease that affects many women during
pregnancy, especially in the third trimester (1).
This disease is now recognized pathophysiologically as an endothelial
dysfunction that probably is related to the accumulation of lipid
peroxides produced in the placenta (2)(3). The
placenta also has opposite physiological activities, including the
production of antioxidants that decompose the placental lipid peroxides
(3)(4)(5). Generally, a healthy pregnancy is maintained by the
quantitative balance between such oxidants and antioxidants
(3)(4)(5), and the disintegration of this equilibrium might
lead to preeclampsia (3)(4)(5). Until now, the kinds of
substances that function as placental antioxidants have been unknown.
Catechol estrogen (free form) has been considered as one of the most
likely candidates, acting as an endogenous antioxidant (6).
However, we suspected the effect of free catechol estrogen
because of its low plasma concentration (7) and rapid
metabolic clearance rate in humans
(7)(8).
Previously, we reported a negative correlation between the serum
concentration of estradiol 17-sulfate
(ES)1
and those of lipid peroxides in late pregnancy (9).
The results led us to speculate that ES is metabolized to its
catechols, which act as lipid peroxide scavengers during pregnancy.
This speculation was based on the following findings: human placental
microsomes have a fairly high 2-hydroxylase activity toward ES
(10), and the product 2-hydroxyestradiol 17-sulfate
(2-OH-ES) has a strong inhibitory effect on lipid peroxidation
(11). More recently, we reported the presence of 2-OH-ES in
the blood of pregnant women and its lower concentrations in the
peripheral blood of pregnant women with pregnancy-induced hypertension,
compared with healthy pregnant women (12). Therefore,
whether 2-OH-ES is present in the urine of pregnant women is of
particular interest.
Here, we describe our discovery of 2-OH-ES in the urine of pregnant
women and its measurement during pregnancy by a previously established
RIA (13). The reliability of the RIA method was confirmed by
HPLC using electrochemical detection.
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Materials and Methods
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reagents
The steroidal conjugates, ES (14), 2-OH-ES
(14), 4-hydroxyestradiol 17-sulfate (4-OH-ES)
(14), 2-methoxyestradiol 17-sulfate (2-OMe-ES)
(14), and [6,7-1
H] 2-OH-ES (1.2
x 1012 Bq/mmol) (13) were prepared as
described previously. Estriol 16-glucuronide (E3-16-G) and dextran
(T-70) were obtained from Sigma. Charcoal (Norit A) was
purchased from Nacalai Tesque. Dextran-coated charcoal was prepared
from 50 g/L charcoal and 5 g/L dextran in assay buffer solution
(15). Bovine serum 
-globulin (Cohn fraction II) and
bovine serum albumin (fraction V) were purchased from Sigma.
Sep-Pak C18 cartridges and Millex 4-mm HV were
purchased from Waters and Millipore, respectively. All other reagents
and solvents were of reagent grade.
subject information
All subjects were outpatients in this study. The procedure
followed was in accordance with the Helsinki Declaration of 1975 as
revised in 1996. The aim of the present study was explained to each
pregnant woman and puerperant, and informed consent was obtained from
all women. The mean ages of the pregnant women in the first,
second, and third trimesters were 30.7 years (range, 23–38 years;
n = 13), 30.4 years (range, 20–40 years; n = 21), and 29.0
years (range, 17–42 years; n = 54), respectively. The mean age of
the puerperants was 28.0 years (range, 21–33 years; n = 10). No
drugs were administered to any of the subjects until urine was
collected.
urine collection
Urine samples were obtained from healthy pregnant and preeclamptic
women, and puerperants at 2–24 h after delivery. Ascorbic
acid was added to the collected urine samples to a final
concentration of 0.1 g/L, and urines were stored at -20 °C
until analyzed. Urinary creatinine was measured by the Folin-Wo method.
ria
The assay was carried out as described previously (13)
except for the method of bound and free separation. To urine samples
(0.2 mL, diluted with distilled water when necessary) or aqueous
2-OH-ES solution (0.2 mL; 0–100 ng) were added the following two
solutions: 0.2 mL of [6,7-1
H] 2-OH-ES (20 000
dpm) in 50 mmol/L Tris-HCl buffer solution (pH 7.0) containing 0.5 g/L
bovine serum -globulin, 0.6 g/L bovine serum albumin, and 0.1 g/L
ascorbic acid; and 0.6 mL of antiserum diluted 1:1000 with the same
buffer solution. The mixture (1 mL) was incubated at 4 °C for
18 h, after which 0.5 mL of a suspension of dextran-coated
charcoal was added. The mixture was then incubated at 4 °C for 10
min, and then centrifuged at 1500g for 10 min to remove the
charcoal. The radioactivity of the duplicate supernatant fractions
(2 x 0.5 mL) was measured by an Aloka LSC-1000 liquid
scintillation spectrophotometer in ACS-II (Amersham) as the
scintillator.
hplc
Apparatus.
HPLC was carried out in a model CCPS (Tosoh)
equipped with an EC-8011 electrochemical detector (Tosoh) at
700 or 900 mV vs a Ag/AgCl reference electrode as described previously
(16). A Mightysil RP-18GP column packed with 5-µm
particles (250 x 3.0 mm i.d.; Kanto) was used as a stationary
phase and maintained at 40 °C in a column heater. Data processing
was performed by a Model C-R6A Chromatopac (Shimadzu).
Separation.
The chromatographic condition for the simultaneous
separation of ES, 2-OH-ES, 4-OH-ES, 2-OMe-ES, and E3-16-G were
established, using various concentrations of 5 g/L
NH4H2PO4
(pH 3.0) and methanol as the mobile phase and a flow rate of 0.4
mL/min.
Calibration curve for 2-OH-ES.
The calibration curve for
2-OH-ES was obtained by injecting a known amount of 2-OH-ES and
plotting the relationship between the injected amount and the peak
area.
Preparation of the 2-OH-ES-free urine.
Sulfate-free urine was
prepared by the method of Heyns et al. (17). Charcoal (50
mg) was added to 1 mL of a pooled urine sample, and the mixture was
stirred vigorously for 30 min at room temperature. The mixture was
centrifuged at 1500g for 10 min to remove the charcoal, and
the supernatant was filtered through a membrane filter to give the
2-OH-ES-free urine.
Recovery of 2-OH-ES.
A known amount of 2-OH-ES was added to
the 2-OH-ES-free urine, and the mixtures were analyzed by HPLC.
comparison of urinary 2-oh-es concentrations measured by ria and
hplc
Urine samples collected from pregnant women and from puerperants
were divided into two fractions, and 1 mL of each fraction was analyzed
by RIA and HPLC as described.
urinary 2-oh-es
Measurement of urinary 2-OH-ES was carried out as follows: A 1-mL
aliquot of each urine sample was passed through a Sep-Pak
C18 cartridge, followed by washing with 2 mL of
distilled water. The steroid-containing fraction was obtained by
elution with methanol (4 mL), and the eluates were evaporated under a
nitrogen stream at 40 °C to give the residue, which was dissolved in
100 µL of methanol containing 0.1 g/L ascorbic acid. After being
passed through a Millex 4-mm HV filter, the solution was subjected to
HPLC.
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Results
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Urinary 2-OH-ES was measured by RIA with a slight modification of
the previous method (13). In constructing the calibration
curve, we used 1000-fold diluted antiserum and plotted the percentage
of labeled 2-OH-ES bound against various amounts of 2-OH-ES. The
calibration curve obtained was linear for 0.05–100 ng of
2-OH-ES. Urinary 2-OH-ES concentrations measured by this
method were expressed as µg/mg creatinine.
In the present report, the HPLC detection and assay method for urinary
2-OH-ES was established to confirm the RIA method. The chromatographic
conditions for quantitative analysis of urinary estrogen conjugates
were established using such authentic conjugates as ES, its potential
ring-A metabolites (2-OH-ES, 4-OH-ES, and 2-OMe-ES), and E3-16-G
(18), whose urinary excretion rate is known to increase
during pregnancy. When a 60:40 (by volume) mixture of 5 g/L
NH4H2PO4
(pH 3.0) and methanol was used as the mobile phase, the above authentic
steroids were separated satisfactorily, as shown in Fig. 1
.
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Figure 1. Electrochemical detection at different voltages of the
authentic estrogen conjugates separated by HPLC.
The dotted lines indicate the much higher peaks heights
for E3-16-G (peak 1) and ES (peak 4) at
900 mV compared with their heights at 700 mV (solid
lines). The other peaks shown are 4-OH-ES
(2) 2-OH-ES (3), and 2-OMe-ES
(5). The injected amount of each conjugate was 40 ng.
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The conditions for sensitive detection of 2-OH-ES were established by
controlling the detector voltage of the electrochemical detector. As
described previously (16), catechol- or
guaiacol-type conjugates could be detected with adequate
sensitivity even in the presence of phenol-type conjugates by
controlling the detector voltage. Fig. 1
shows the results obtained,
where solid and dotted lines represent the chromatograms detected at
700 and 900 mV, respectively. There were almost no differences in the
peak heights of the catechol- or guaiacol-type conjugates detected at
700 or 900 mV. In sharp contrast, when the voltage was lowered from 900
to 700 mV, the peak heights of phenol-type conjugates were
substantially reduced or not detectable. These results indicated that
sensitive analysis of 2-OH-ES could be accomplished by decreasing or
eliminating the influence of the large amounts of the phenol-type
conjugates present in pregnancy urine.
A representative HPLC chromatogram of the estrogen-containing fraction
from pregnancy urine, which was obtained at the detection voltage of
700 mV, is shown in Fig. 2
. Peak assignment in the chromatogram was achieved by the
addition of authentic conjugates to the sample. The lowering of the
voltage from 900 to 700 mV reduced the size of the E3-16-G peak
substantially, allowing the discrimination of small peaks of catechol-
or guaiacol-type conjugates.
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Figure 2. HPLC of the estrogen-containing fraction from pregnancy
urine (the third trimester) at a detection voltage of 700 mV.
Peak numbers correspond to those in the legend for Fig. 1
.
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The calibration curve was constructed by plotting the peak area against
the amount of 2-OH-ES injected, and satisfactory linearity was observed
for 2-OH-ES concentrations of 2–200 ng (y =
1.14x + 0.014; r = 0.998). To
confirm the validity of the method for the determination of urinary
2-OH-ES, a known amount of authentic 2-OH-ES was added to the
sulfate-free urine, and the conjugate recovered through the whole
clean-up procedure was determined. It is evident from the data in Table 1
that 2-OH-ES was recovered to a satisfactory extent.
Once the analytical method of urinary 2-OH-ES by HPLC was
satisfactorily established, the reliability of the RIA method was
verified. The urine sample was divided into two parts, each of which
was analyzed by RIA and HPLC. As indicated in Fig. 3
, the urinary 2-OH-ES values obtained by RIA showed a close
relationship to those obtained by HPLC; the equation in the regression
line was: y = 1.10x - 0.0143
(r = 0.958; n = 35; P <0.01). The RIA
method thus was demonstrated to be useful. Because of its simplicity,
ease of use, and capacity for handling numerous samples, the
measurement of 2-OH-ES in the urine from pregnant women or puerperants
was carried out by the RIA method.
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Figure 3. Relationship between urinary 2-OH-ES values obtained by
RIA and by HPLC.
Equation for the regression: y =
1.10x - 0.0143; r = 0.958;
P <0.01; n = 35.
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The urinary 2-OH-ES concentrations of pregnant women during gestation
are shown in Fig. 4
, where circles (
) and triangles (
) indicate healthy
pregnant women and preeclamptic women, respectively. The urinary
2-OH-ES concentrations of healthy pregnant women increased as the
gestation progressed; the concentrations in the first, second, and
third trimesters were 2.0 ± 0.6 (mean ± SE; n = 13),
5.3 ± 1.3 (n = 21), and 15.3 ± 2.0 (n = 54)
µg/mg creatinine, respectively. The concentrations during the third
trimester were seven- and threefold higher than those during the first
and second trimester, respectively. The concentration in the
preeclampsia cases was 3.9 ± 1.9 µg/mg creatinine (mean ±
SE; n = 12), which is approximately one-fourth the value in
healthy cases in the same stage. The differences in urinary 2-OH-ES
concentrations between the first and second trimesters, and the second
and third trimesters were significant, at P <0.05 and
P <0.01, respectively. The difference in the urinary
concentrations between healthy pregnant women and preeclamptic women
during the third trimester was also significant, at P
<0.01. The 2-OH-ES concentrations at 2–24 h after delivery decreased
to <0.15 µg/mg creatinine (n = 10); this value was irrelevant
to the clinical history of pregnant women with or without preeclampsia.
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Figure 4. Urinary 2-OH-ES concentrations during pregnancy and the
comparison in the third trimester between healthy pregnancy and
preeclampsia.
, healthy pregnant women; , preeclamptic women. Horizontal
bars indicate the mean urinary 2-OH-ES concentrations for each
group. *, P <0.05, comparison between the first and
second trimesters; **, P <0.01, comparison between
the second and third trimesters; ***, P <0.01,
comparison between healthy pregnancy and preeclampsia in the third
trimester.
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Discussion
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In the present study, it became evident that 2-OH-ES is excreted
into the urine of healthy pregnant women in fairly high concentrations.
As far as we know, this is the first report describing the detection of
this estrogen sulfate in human urine. In addition, we demonstrated that
urinary 2-OH-ES increases as gestation progressed, which is considered
quite natural because of similar evidence that urinary ES increases
with the progress of gestation (19), and because of the high
2-hydroxylase activity toward ES by human placental microsomes
(10). Our recent report (12) dealing with the
existence of this sulfate in the serum of pregnant women also supports
the present results.
As reported previously, the antiserum used in this assay was highly
specific for 2-OH-ES, and its cross-reactivities with similar estrogen
conjugates were extremely low (13). For example, the
cross-reactivities of such similar conjugates as ES, 2-OMe-ES, and
4-OH-ES, were 0.1%, 0.03%, and 0.01%, respectively. Even E3-16-G
(18), excreted in large quantity into the urine during
pregnancy, showed a cross-reactivity <0.01%. However, there still
remains the possibility of the presence of unknown urinary substance(s)
with high cross-reactivity to the antibody. To ensure that the above
RIA is applicable to the measurement of urinary 2-OH-ES, we developed
the HPLC method for the same urine samples.
As shown in Fig. 3
, urinary 2-OH-ES values obtained by RIA exhibited a
close correlation to those obtained by HPLC. The RIA established thus
is reliable and applicable to the urine samples. The measurement of
urinary 2-OH-ES was carried out by RIA because it was superior to HPLC
in its ability to deal with a large number of samples.
In Fig. 4
, urinary 2-OH-ES is shown to increase as gestation
progresses, especially in the third trimester. Although the
concentrations seem to be uneven among individuals, the urinary 2-OH-ES
concentration (15.3 ± 2.0 µg/mg creatinine) in the third
trimester is significantly higher than in the first two
trimesters, and is ~370-fold higher than the urinary ES
(41.4 ± 4.7 ng/mg creatinine) at the same stage, as reported
previously (19). The big difference between the urinary
concentrations of ES and 2-OH-ES implies the conversion of ES to
2-OH-ES during pregnancy, especially in the last stages of the
pregnancy. This can be explained by the presence of high
2-hydroxylase activity toward ES in the placental microsomes
(10). These results suggest the physiological requirement of
this aromatic hydroxylation during pregnancy.
As candidate substance for the placental antioxidants, there have been
several reports nominating superoxide dismutase (20)(21)(22),
catalase (21)(22), glutathione
peroxidase (21)(22), or catechol
estrogens (6). Of these, superoxide dismutase, catalase, and
glutathione peroxidase are not now recognized as participating actively
in the maintenance of healthy pregnancy because there seem to be no
increasing tendencies in their production with the progression of
gestation (22). Catechol estrogens such as 2-hydroxyestrone
or 2-hydroxyestradiol have recently attracted considerable attention as
the most possible candidates, especially because of their strong
antioxidant effect (23). However, it seems that some
inexplicable problems remain in the assignment of catechol estrogens as
placental antioxidants. Plasma circulating catechol estrogens are known
to be rapidly metabolized by erythrocytic catechol
O-methyltransferase to the corresponding guaiacol estrogens,
such as 2-methoxyestrone or 2-methoxyestradiol
(7)(8). The plasma concentration of catechol
estrogens thus is extremely low (7). Although guaiacol
estrogens evidently have a strong antioxidant effect (11),
we cannot consider them as placental antioxidants because they bind
strongly or irreversibly to plasma testosterone-estradiol binding
globulin (24).
In contrast to free-formed catechol estrogens, the
C17-sulfoconjugated estrogen (2-OH-ES) has some
properties that may make it advantageous as a placental
antioxidant: (a) the 2-hydroxylase activity of
placental microsomes is higher toward ES than estradiol
(10); (b) the plasma metabolic clearance rate of
2-OH-ES is lower than that of 2-OH-E in rats (25);
(c) 2-OH-ES has a strong antagonistic effect against lipid
peroxidation (11); and (d) 2-OH-ES concentrations
are significantly higher in umbilical arteries than in the maternal
peripheral vein (12). In addition, the two results observed
in the present study—the rapid decrease in urinary 2-OH-ES after
delivery, and the lower urinary 2-OH-ES concentrations in preeclampsia
compared with healthy pregnancy—may support our hypothesis.
In conclusion, 2-OH-ES may be clinically important in the field of
obstetrics. In this report, urinary 2-OH-ES was measured directly by
RIA without hydrolysis, the methodology was shown to be simple and
easy, and the reliability of the method was supported by comparison
with another method (HPLC). Further detailed studies, such as
a follow-up measurement of urinary 2-OH-ES in individual subjects, or
an investigation of the effect of medical treatment on urinary 2-OH-ES
in preeclamptic women, may provide additional evidence of its
usefulness as a marker for the diagnosis of preeclampsia. The RIA
established here may become a powerful tool in the development of this
study.
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Acknowledgments
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This work was supported in part by a Grant-in-Aid for Encouragement
of Young Scientists from the Ministry of Education (1996, K. Takanashi)
and the Akiyama Research Grant from The Akiyama Foundation (1998, K.
Takanashi).
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Footnotes
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1 Nonstandard abbreviations: ES, estradiol 17-sulfate; 2-OH-ES, 2-hydroxyestradiol 17-sulfate; 4-OH-ES, 4-hydroxyestradiol 17-sulfate; 2-OMe-ES, 2-methoxyestradiol 17-sulfate; and E3-16-G, estriol 16-glucuronide. 
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Abstract
Introduction
