Fetal-to-maternal transfer of 3,3',5-triiodothyronine sulfate and its metabolite in sheep

Sing-Yung Wu1, Daniel H. Polk2, Wen-Sheng Huang3, and Delbert A. Fisher2

1 Nuclear Medicine and Medical Services, Department of Veterans Affairs Medical Center, Long Beach 90822; 2 Perinatal Laboratory, Harbor-University of California Los Angeles Medical Center, Torrance, California 90509; and 3 Department of Nuclear Medicine, Tri-Service General Hospital, Taipei, Taiwan 107, China


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -globulin (the second antibody) was purchased from Calbiochem (La Jolla, CA).

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).

                              
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Table 1.   Changes of serum and urinary concentrations of T2S, T3S, and T3 in fetal and maternal sheep after acute T3S infusion into fetuses


                              
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Table 2.   Changes of serum and urinary concentrations of T2S, T3S, and T3 in fetal and maternal sheep after acute T3S infusion into ewes


                              
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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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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Fig. 1.   Changes of serum 3,3'-diiodothyronine sulfate (T2S; A) and 3,3',5-triiodothyronine (T3) sulfate (T3S; B) concentrations in maternal ewes of 130-day gestation after a bolus injection of 300 µg T3 (black-triangle, solid lines) or 340 µg T3S (open circle , broken lines) to fetuses. * P < 0.05; ** P < 0.01 vs. baseline values.

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 congruent 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Chopra, I. J., R. S. Ho, and R. Lam. An improved radioimmunoassay for measurement of triiodothyronine in human serum. J. Lab. Clin. Med. 80: 729-739, 1972[Medline].

2.   Chopra, I. J., S. Y. Wu, G. N. Chua Teco, and F. Santini. A radioimmunoassay for measurement of 3,5,3'-triiodothyronine sulfate studies in thyroidal and non-thyroidal disease, pregnancy, and neonatal life. J. Clin. Endocrinol. Metab. 75: 189-194, 1992[Abstract].

3.   Eelkman Rooda, S. J., E. Kaptein, M. A. C. van Loon, and T. J. Visser. Development of a radioimmunoassay for triiodothyronine sulfate. J. Immunoassay 9: 125-134, 1988[Medline].

4.   Galton, V. A., E. Martinez, A. Hernandez, E. A. St. Germain, J. M. Bates, and D. L. St. Germain. Pregnant rat uterus expresses high levels of the type 3 iodothyronine deiodinase. J. Clin. Invest. 103: 979-987, 1999[Abstract/Free Full Text].

5.   Hobkirk, R., and M. A. Glasier. Estrogen sulfotransferase distribution in tissues of mouse and guinea pig: steroidal inhibition of the guinea pig enzyme. Biochem. Cell Biol. 70: 712-715, 1992[Medline].

6.   Hurd, R. E., F. Santini, B. Lee, P. Naim, and I. J. Chopra. A study of the 3,5,3'-triiodothyronine sulfation activity in the adult and fetal rat. Endocrinology 133: 1951-1955, 1993[Abstract].

7.   Kirk, R. E. Experimental Design (2nd ed.). Belmont, CA: Brooks-Cole, 1982, p. 112-114.

8.   Mol, J. A., and T. J. Visser. Synthesis and some properties of sulfate esters and sulfamates of iodothyronines. Endocrinology 117: 1-7, 1985[Abstract].

9.   Polk, D. H., D. A. Fisher, and S. Y. Wu. Alternate pathways of thyroid hormone metabolism in developing mammals. In: Thyroid Hormone Metabolism: Molecular Biology and Alternate Pathways, edited by S. Y. Wu, and T. J. Visser. Boca Raton, FL: CRC, 1994, p. 223-243.

10.   Polk, D. H., A. Reviczky, S.-Y. Wu, W.-S. Huang, and D. A. Fisher. Metabolism of sulfoconjugated thyroid hormone derivatives in developing sheep. Am. J. Physiol. 266 (Endocrinol. Metab. 29): E892-E895, 1994[Abstract/Free Full Text].

11.   Visser, T. J. Sulfation and glucuronidation pathways of thyroid hormone metabolism. In: Thyroid Hormone Metabolism: Molecular Biology and Alternate Pathways, edited by S. Y. Wu, and T. J. Visser. Boca Raton, FL: CRC, 1994, p. 85-117.

12.  Wu, S. Y., D. A. Fisher, G. Jones, W. H. Florsheim, D. L. St. Germain, and V. A. Galton. Characterization of a novel iodothyronine sulfotransferase activity in pregnant rat uterus (Abstract). Program 81st Annual Meeting of the Endocrine Society San Diego 1999.

13.   Wu, S. Y., D. A. Fisher, D. Polk, and I. J. Chopra. Maturation of thyroid hormone metabolism. In: Thyroid Hormone Metabolism, Regulation and Clinical Implications, edited by S. Y. Wu. Boston, MA: Blackwell, 1991, p. 293-320.

14.   Wu, S. Y., W. S. Huang, D. Polk, W. L. Chen, A. Reviczky, J. Williams, I. J. Chopra, and D. A. Fisher. The development of a radioimmunoassay for reverse-triiodothyronine sulfate (rT3S) in human serum and amniotic fluid. J. Clin. Endocrinol. Metab. 76: 1625-1630, 1993[Abstract].

15.   Wu, S. Y., W. S. Huang, D. Polk, W. H. Florsheim, W. L. Green, and D. A. Fisher. Identification of thyroxine-sulfate (T4S) in human serum and amniotic fluid by a novel T4S radioimmunoassay. Thyroid 2: 101-105, 1992[Medline].

16.   Wu, S. Y., D. H. Polk, W. L. Chen, D. A. Fisher, W. S. Huang, and B. Yee. A 3,3'-diiodothyronine sulfate cross-reactive compound in serum from pregnant women. J. Clin. Endocrinol. Metab. 78: 1505-1509, 1994[Abstract].

17.   Wu, S. Y., D. H. Polk, D. A. Fisher, W. S. Huang, P. Beck-Peccoz, C. H. Emerson, S. W. Kuo, and W. L. Chen. Urinary Compound W in pregnant women is a potential marker for fetal thyroid function. Am. J. Obstet. Gynecol. 178: 886-891, 1998[Medline].

18.   Wu, S.-Y., D. H. Polk, D. A. Fisher, W.-S. Huang, A. L. Reviczky, and W.-L. Chen. Identification of 3,3'-T2S as a fetal thyroid hormone derivative in maternal urine in sheep. Am. J. Physiol. 268 (Endocrinol. Metab. 31): E33-E39, 1995[Abstract/Free Full Text].

19.   Wu, S.-Y., D. H. Polk, W.-S. Huang, A. Reviczky, K. Wang, and D. A. Fisher. Sulfate conjugates of iodothyronines in developing sheep: effect of fetal hypothyroidism. Am. J. Physiol. 265 (Endocrinol. Metab. 28): E115-E120, 1993[Abstract/Free Full Text].

20.   Wu, S. Y., D. H. Polk, S. Wong, A. Reviczky, R. Vu, and D. A. Fisher. Thyroxine sulfate is a major thyroid hormone metabolite and a potential intermediate in the monodeiodination pathways in fetal sheep. Endocrinology 131: 1751-1756, 1992[Abstract].


Am J Physiol Endocrinol Metab 277(5):E915-E919
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