1 Nuclear Medicine and Medical
Services, Earlier studies have shown that sulfoconjugation
is a major pathway of thyroid hormone metabolism in fetal mammals. To
assess the placental transfer of sulfoconjugates in the pregnant sheep model, we measured 3,3',5-triiodothyronine
(T3) sulfate
(T3S), 3,3'-diiodothyronine
sulfate (T2S), and
T3 concentrations in fetal serum
and in maternal serum and urine after
T3S infusion to the fetus
(n = 5) or the ewe
(n = 6). Maternal infusion of
T3S did not increase fetal serum
T2S,
T3S, or
T3 concentrations. In contrast, fetal infusion of T3S produced
significant increases in maternal serum
T2S and
T3S but not
T3 concentrations. Fetal
T3S infusion also increased
maternal urine excretion of T3S.
However, the 4-h cumulative maternal urinary excretion of
T2S and
T3S after fetal T3S infusion was less than the
excretion observed after fetal infusion of equimolar amounts of
T3 in our previous study. It is
concluded that fetal serum T2S and
T3S can be transferred to maternal
compartments. However, compared with
T3, these sulfoconjugates may be
less readily transferred.
sulfoconjugate; thyroid hormone metabolism
PREVIOUS STUDIES of thyroid hormone metabolism in the
ovine, rat, and human fetus have documented persistently low serum
3,3',5-triiodothyronine (T3) concentrations (13). This
is partly the result of low tissue type I 5'-iodothyronine
monodeiodinase (D1) activity, leading to low rates of monodeiodination
of thyroxine (T4) to
T3. The low fetal tissue D1
activity also decreases the clearance of reverse T3
(rT3), so that circulating
rT3 levels in the fetus, relative to adult animals, are increased. In addition, activities of type III
5-iodothyronine monodeiodinase are relatively high in fetal tissues and
in the placenta, so that production rates of
rT3 from T4 are increased in the fetus
compared with the adult (13). More recently, we have demonstrated
sulfoconjugates of T4
(T4S), T3
(T3S), reverse
T3
(rT3S), and,
3,3'-diiodothyronine (T2S)
in significant concentrations in biological fluids of sheep and human fetuses (2, 9, 13-16, 19, 20). Kinetic studies in third trimester
fetal sheep have shown, in contrast to adult animals, that daily
production rates of T4S,
T3S, and
rT3S far exceed production of
T3 and
rT3 (10). Thus the pathways of
thyroid hormone metabolism in the fetus differ from the adult in that
T4S,
rT3S, and
T3S are the predominant
metabolites. This is likely due to the low levels of D1 activities in
fetal tissues (9, 10).
Infusion of T3 to the fetal sheep
rapidly increases maternal urinary excretion of
T2S and
T3S in the absence of significant changes in maternal serum T3
concentrations, whereas maternal T3 infusion produces a lesser
increase in urinary T2S and
T3S excretion relative to serum
T3 concentrations (18). We
concluded from these earlier studies that ovine fetal
T3 infusion produces significant
fetal-to-maternal transfer of T2S
and T3S and suggested that
transferred sulfated iodothyronine in maternal serum and urine could be
used as a noninvasive marker for fetal thyroid function (16). Indeed,
in humans, but not in other laboratory mammals, we did find high
concentrations of a T2S-like
material (Compound W) in the serum and urine of pregnant women (16,
17). To further characterize fetal-to-maternal and maternal-to-fetal transfer of T2S and
T3S, we have conducted fetal and
maternal infusions of T3 and
T3S in the pregnant sheep model.
In this report, we present the T3S
infusion results and compare these with our earlier
T3 study.
T3,
T3S, and
T2S RIAs.
T3,
T3S, and
T2S levels in
serum and urine were measured by specific and sensitive RIAs (1, 2, 18). Serum and urine samples were extracted with 2 vol of 95% ethanol
(final ethanol concentration, 63%) as described previously (2, 16,
18). T2S RIA has a lower limit of
detection of 2 pg (3.3 fmol) or 2 ng/dl. Of various thyroid hormone
analogs studied and known to exist in sheep serum or urine, only
T3S, rT3S, and
T4S cross-react significantly
(3.2, 1.4, and 0.02%, respectively) in the
T2S RIA;
T4,
T3,
rT3, and
T2 cross-reacted <0.0001%. The
T2S concentrations in serum and
urine were corrected for the cross-reactivity of
T3S. The
T3S RIA has a lower limit of
detection of 2 ng/dl (2.7 fmol). Analog cross-reactivities in the
T3S RIA are:
T4, <0.001;
rT3, <0.001;
T3, <0.001;
rT3S, <0.007; and
T4S, 3.3%.
Animal preparation and samples.
Western mixed-bred, time-dated pregnant ewes with singleton pregnancies
were obtained at 121 ± 2 days (term is ~150 days) from the
Nebeker Ranch (Lancaster, CA) and acclimated to our laboratory
conditions and food. Fetal catheterization was accomplished with
previously reported techniques (18). After the ewes recovered from
surgery (4-5 days), 0.46 µmol of
T3S (340 µg) was infused into
the fetus by a Harvard syringe pump (Halliston, MA) over a period of
3-5 min. In addition, on separate days, 2.3 µmol of
T3S (1,700 µg) were infused over
3-5 min into the maternal circulation. Fetal and maternal serum
and maternal urine samples were collected hourly for 5 h.
These T3S doses were comparable to
T3 doses selected in the infusion
study in fetal and maternal sheep reported previously (18). All animal protocols were reviewed and approved by our institutional animal use
committee in accordance with American Association of Laboratory Animal
Care Guidelines.
Sources of materials.
T2 and
T3 were purchased from
Henning-Berlin (Berlin, Germany).
T2S,
T3S,
125I-T2S,
and
125I-T3S
were prepared by the method of Eelkman Rooda et al. (3, 8).
Chlorosulfonic acid, 99%, was purchased from Aldrich Chemical (Milwaukee, WI). The final purification of
T2S and
T3S was made by reversed-phase
HPLC with a preparative column (Biochrom 1010 ODS; Regis, Morton Grove,
IL). The products were eluted isocratically with a mixture of
acetonitrile and 20 mM ammonium acetate, pH 4.0 (22:78 vol/vol), at a
solvent flow rate of 10 ml/min.
T2S and
T3S were recovered with purity
>99%, as assessed by HPLC. T3S
purity was 95% as assessed by T3S
and T3 RIAs. Goat anti-rabbit Statistical analysis. ANOVA was used
for multigroup comparisons. If significant differences were detected,
Dunnett's multicomparison test was used to compare the control or
baseline mean and the mean values of other groups (7). Significance was
defined as P < 0.05. Results are
reported as means ± SE.
T2S,
T3S, and
T3 concentration in maternal
serum and urine after fetal infusion of
T3S. After bolus
infusion of the 130 ± 3-day gestation ovine fetus with 0.46 µmol
of T3S (340 µg), hourly fetal
serum and maternal urine and serum samples were collected. The
concentrations of T2S,
T3S, and
T3 in the fetuses and ewes are
shown in Tables 1 and
2. Excretion of
T2S,
T3S, and
T3 over 4 h in maternal urine, in
comparison with excretion after infusion of an equimolar dose of
T3 reported previously (18), are
shown in Table 3. At 1 h,
there were marked increases in fetal serum T2S,
T3S, and
T3 concentrations (Table 1).
Maternal serum T3 remained
unchanged, but there were significant increases in maternal serum
T2S and
T3S levels from 2 to 4 h. Over
4 h, the cumulative excretions of
T2S,
T3S, and
T3 in maternal urine after bolus
T3S infusion represented 0.18, 0.18, and 0.026%, respectively, of the injected dose compared with
0.44, 0.51, and 0.044%, respectively, after
T3 infusion in our earlier study
(Ref. 18 and Table 3).
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-globulin (the second antibody) was purchased from Calbiochem (La
Jolla, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Table 1.
Changes of serum and urinary concentrations of T2S,
T3S, and T3 in fetal and maternal sheep
after acute T3S infusion into fetuses
Table 2.
Changes of serum and urinary concentrations of T2S,
T3S, and T3 in fetal and maternal sheep
after acute T3S infusion into ewes
Table 3.
Maternal excretion of T2S, T3S, and
T3 after fetal or maternal T3S infusion
compared with T3 infusion reported previously (18)
After bolus infusion of T3S (2.3 µmol) into the maternal ewes at 130 ± 3 days, there were marked increases in maternal serum T2S, T3S, and T3 (Table 2). However, there were minimal and statistically insignificant increases in fetal serum concentrations of T2S, T3S, and T3 (Table 2). The 4-h excretion of T3S was 11.5% in maternal urine after T3S infusion, an amount significantly higher than that after T3 infusion (0.61%; Table 3).
Significantly marked increases in serum T3 concentrations in both fetuses and ewes were observed after respective fetal and maternal T3S infusions (Table 1 and 2). The average molar ratio of serum T3 to T3S concentrations in fetuses after fetal T3S infusion was 0.067% at 1 h decreasing to 0.029% at 5 h. The average molar ratios of serum T3 to T3S concentrations in ewes after maternal T3S infusion were 0.348 at 1 h and 0.816 at 5 h. Similar increases in the average molar ratios of maternal urinary T3 to T3S concentrations were also observed after maternal T3S infusion, 0.01 at 1 h and 0.09 at 5 h (Table 2).
Also, there were marked increases in serum T2S levels in fetuses after fetal T3S infusion in contrast to relatively small increases in maternal serum T2S values after maternal T3S infusion (Tables 1 and 2). The average molar ratio of fetal serum T2S to T3S concentration was 0.037 at 1, increasing to 0.10 at 5 h.
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DISCUSSION |
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In our previous study, a fetal bolus infusion of
T3 immediately increased maternal
serum and urine concentrations of
T2S and T3S, whereas maternal serum
T3 concentrations remained
unchanged. Maternal infusion of T3
increased serum and urine T2S and
T3S levels, but relative to
T3, the values were much less than
those measured after fetal T3
infusion. We concluded that fetal
T2S and
T3S readily cross the placenta to
appear in maternal serum and urine. In the present study, we infused
T3S in molar equivalent amounts
into both fetuses and ewes to compare results with the previous
T3 infusion data. Maternal serum
T2S and
T3S concentrations measured after
fetal infusion of equimolar doses of
T3 in the earlier study or
T3S in the present study are shown
in Fig. 1. Both maternal
T2S and
T3S levels increased after either
fetal T3 or
T3S infusion. However, maternal
T2S and
T3S concentrations were
significantly higher after fetal
T3 infusion than after
T3S administration despite the
fact that the mean fetal serum concentration of
T3S after fetal
T3S infusion was 20 times higher
than T3S concentrations observed
after fetal T3 infusion (in the
previous study) and remained some 10 times higher than in
T3-infused fetuses at the end of 4 h (Ref. 18; Tables 1 and 2). Thus it appears that
T3 rather than
T3S is the major fetal precursor
for the T2S and
T3S that appear in the maternal
compartment.
|
The site(s) of monodeiodination of T3 to T2 and of sulfation of T3 and T2 remains unclear. Placental type III iodothyronine deiodination activity deiodinates T3 to T2, whereas the sulfated iodothyronines are not substrates for placental type III or type II deiodinase (11). Galton et al. (4) have demonstrated the presence of type III deiodinase in rat uterus, suggesting that this tissue also could be involved in the fetal-to-maternal T3 exchange. However, the site(s) of sulfation remains unclear. Fetal tissues would be excluded because fetal T3S infusion does not increase maternal T3S as effectively as fetal T3 infusion. Placental and/or uterine sulfotransferase would appear to be involved in facilitating fetal-to-maternal transfer of T3 and T2. Sulfotransferase activity has been demonstrated in rat placenta, but the levels were low (6). Mouse, human, and sheep placentas have been shown to sulfate estrogens efficiently (5, 6). More recently, we demonstrated significant iodothyronine sulfotransferase activity in rat uterus (12) and sheep cotyledon (Wu and Fisher, unpublished observation), suggesting that the uterus/placenta may indeed play a role in facilitating fetal-to-maternal transfer of T3 and T2.
The marked early increase of serum T3 concentrations in the fetus after T3S infusion is partly due to impurity of T3S, which had a T3-to-T3S molar ratio of 0.05 measured by T3 and T3S RIAs. However, the similar molar ratios of serum T3 to T3S concentrations at 1 and 4 h (0.067 and 0.055, respectively) in fetuses after T3S infusion suggest that the clearance rates of T3 and T3S in ovine fetuses were similar. By contrast, the molar ratio of serum T3 to T3S concentrations in ewes after T3S infusion were much higher, 0.326 at 30 min and 0.348 at 1 h, increasing to 0.816 at 5 h. These data suggest that significant T3S desulfation occurs in ewes but is minimal in fetuses. The continuing increase of the serum T3-to-T3S molar ratio in ewes also may reflect rapid urinary clearance of T3S in ewes as shown in Table 3.
The marked and significant increases in serum T2S concentrations in fetuses after fetal T3S infusion contrast with the minimal increases in T2S levels in maternal serum after maternal T3S infusion. This marked and more prolonged elevation of fetal T2S concentration, relative to T3S, suggests that there is significant conversion of T3S to T2S in the fetus. In addition, the further slower metabolism of T2S by type I monodeiodinase probably also contributes (9, 13).
As shown in Table 3, the total amount of excretion of
T3,
T3S, and
T2S in the maternal urine was
0.4% of T3S infused into the
ovine fetus. However, other metabolites, i.e.,
3,3'-T2,
3'-monoiodothyronine (3'-T1),
3'-T1S,
3-T1,
3-T1S, and thyronine, were not
measured due to the limitation of RIA and sample
availability. Furthermore, any fetal-to-maternal
transferred product(s) converted to compound(s) with a longer
biological half-life, such as a
T2S-like material (Compound W)
found in humans, may be accumulated in maternal circulation and could
serve as a useful marker for fetal thyroid function. Whether or not any
physiological role is being played by the transferred metabolite(s)
(such as Compound W) remains to be elucidated.
In summary, the present results, with our earlier T3 infusion studies, demonstrate a significant fetal-to-maternal transfer of T2S and T3S after bolus infusions of T3 or T3S in sheep fetuses of 130 days gestation age. However, the maternal serum levels of T2S and T3S were significantly higher after fetal T3 infusion than those after T3S infusion, and cumulative T2S and T3S excretions into maternal urine also were greater after bolus T3 than after equimolar T3S infusion in fetuses. It is thus concluded that T3, rather than T3S, is the major precursor of the T2S and T3S transferred to maternal ewes. In addition, our results demonstrate more effective desulfation of T3S to T3 in ewes, relative to fetal sheep, and significant T3S-to-T2S conversion in fetal lambs. Further kinetic studies are required to characterize and quantitate fetal-to-maternal transplacental and/or transuterine transfer of sulfated iodothyronines and the physiological significance of such transfer.
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ACKNOWLEDGEMENTS |
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This work has been supported by the Department of Veterans Affairs, National Institutes of Health Grants R15-GM-41949 and HD-04270, and the National Science Council, (ROC) NSC 82-0412-B-016-085.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S.-Y. Wu, Nuclear Medicine and Medical Services (151), VA-UCI Medical Center, 5901 E. 7th St., Long Beach, CA 90822 (E-mail: sywu{at}pop.long-beach.va.gov).
Received 28 January 1999; accepted in final form 18 June 1999.
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