A 3,3’-Diiodothyronine Sulfate Cross-Reactive Material (Compound W), a Potential Marker for Fetal Hypothyroidism

In developing mammals (including humans), a deficiency or excess of thyroid hormone (TH) in the developing brain during the fetal and neonatal periods can lead to morphological and functional abnormalities (Nunez et al., 1992; Pasquini & Adamo, 1994; Morreale de Escobar et al., 2004; Harakawa et al., 1989). The most severe form of hypothyroidism in human fetus and neonate is the syndrome of cretinism. Recent studies indicate that abnormalities in intelligence quotient (IQ) and other neuropsychological tests may be found in children of women with subclinical hypothyroidism during pregnancy (Haddow et al., 1999; Mitchell & Klein, 2004; Casey et al., 2005). Subtle health impairment and reduced socio-educational achievement are observed in young adults with congenital hypothyroidism identified by neonatal screening and subsequently treated (Leger et al., 2011). This suggests the inadequacy in the current strategy with neonatal screening – it may already be too late to secure normal neurological development. Studies of populations indicate that congenital hypothyroidism affects 1 in 3,000 to 4,000 newborns; twice as many females as males. A recent report showed that the incidence in the United States increased from ~ 1:4100 in 1987 to ~ 1:2400 in 2002 (Rastogi & LaFranchi, 2010; Harris & Pass, 2007). The causes for this change are not entirely clear even though the screening strategy may have contributed to the increase (Rastogi & LaFranchi, 2010).


Introduction
In developing mammals (including humans), a deficiency or excess of thyroid hormone (TH) in the developing brain during the fetal and neonatal periods can lead to morphological and functional abnormalities (Nunez et al., 1992;Pasquini & Adamo, 1994;Morreale de Escobar et al., 2004;Harakawa et al., 1989). The most severe form of hypothyroidism in human fetus and neonate is the syndrome of cretinism. Recent studies indicate that abnormalities in intelligence quotient (IQ) and other neuropsychological tests may be found in children of women with subclinical hypothyroidism during pregnancy (Haddow et al., 1999;Mitchell & Klein, 2004;Casey et al., 2005). Subtle health impairment and reduced socio-educational achievement are observed in young adults with congenital hypothyroidism identified by neonatal screening and subsequently treated (Leger et al., 2011). This suggests the inadequacy in the current strategy with neonatal screening -it may already be too late to secure normal neurological development. Studies of populations indicate that congenital hypothyroidism affects 1 in 3,000 to 4,000 newborns; twice as many females as males. A recent report showed that the incidence in the United States increased from ~ 1:4100 in 1987 to ~ 1:2400 in 2002 (Rastogi & LaFranchi, 2010;Harris & Pass, 2007). The causes for this change are not entirely clear even though the screening strategy may have contributed to the increase (Rastogi & LaFranchi, 2010).

Etiology of congenital hypothyroidism
Most cases of congenital hypothyroidism are sporadic. But an estimated 15 to 20 percent of cases are inherited including mutations in the DUOX2, PAX8, SLC5A5(NIS), SLC26A4(PDS), TG, TPO, DEHAL1, TSHB, and TSHR genes (Table 1; Rastogi & LaFranchi, 2010;Grasberger & Refetoff, 2011). Many inherited cases are autosomal recessive but those with a mutation in the PAX8 gene or certain TSHR gene mutations have an autosomal dominant pattern of inheritance. Another possible cause of fetal hypothyroidism is anti-thyroid medication treatment for maternal hyperthyroidism during pregnancy (Rastogi & LaFranchi, 2010;Rovet et al., 1999;Mirabella et al., 2000;Vanmiddlesworth et al., 2011). In severe iodinedeficient regions, the prevalence of cretinism can reach 10% of the local population; WHO estimates indicate a prevalence in the millions worldwide (World Health Organization [WHO], 2004;WHO et al., 2007). Concerns regarding fetal hypothyroidism and timely treatment warrant close monitoring of fetal thyroid status. One method, cordocentesis, is invasive and has a fetal loss rate of 0.5 to 1% (Daffos, 1989). Recently, ultrasonography has been utilized to assess the fetal thyroid gland (Harris & Pass, 2007;Polak & Van Vliet, 2010;Luton et al., 2005;Abuhamad et al., 1995;Ribault et al., 2009). Cordocentesis and serial ultrasonographic measurements are either invasive or laborious. The development of a convenient non-invasive means to monitor fetal thyroid function is definitely needed.

Alternate pathways of Iodothyronines
The alternate pathways of thyroid hormone metabolism include conjugation (sulfation or sulfonation, and glucuronidation of the phenolic hydroxy group) and, to a lesser extent, oxidative deamination of the alanine side-chain leading to the formation of the corresponding iodothyroacetates and ether link cleavage ( Fig. 1; Wu et al., 2005). The alternate pathways of thyroid hormone metabolism include conjugation (sulfation or sulfonation, and glucuronidation) and oxidative deamination of the alanine side-chain leading to the formation of the corresponding iodothyroacetates and ether link cleavage. DIT: diiodotyrosine; tetrac: tetraiodothyroacetic acid; tetram: tetraiodothyronamine.
Alternate pathways may serve as mechanisms for further regulation of the bioavailabilities of thyroid hormones in tissues, in addition to deiodination in various physiological and pathophysiological states. Sulfoconjugation of iodothyronines, for example, is an important pathway in developing mammals ; and sulfated iodothyronines can also be deiodinated, even at a faster rate. Likewise, iodothyroacetates can be sulfated and further deiodinated. Iodothyronine glucuronides are rapidly excreted in the bile. Furthermore, sulfoconjugation and glucuronidation are not irreversible pathways for thyroid hormone metabolism. Glucuronides can be hydrolyzed in the intestine and reabsorbed, and sulfoconjugates can be desulfated in selective tissues, e.g. liver and brain, and become available to nuclear receptors, especially in fetuses where type I deiodinase (D1) activity is low. The in vivo occurrence of the decarboxylated metabolites of T 4 and T 3 , 3,3',5,5'-tetraiodothyronamine (T 4 AM), and 3,3'5-triiodothyronamine (T 3 AM) have not been demonstrated (Leonard & Kohrle, 2000). However, recently, 3-monoiodothyronamine (3monoam or 3-T 1 AM) and thyronamine (T 0 AM) have been identified in brain and other tissues in rodents (Scanlan et al., 2004); monoam was found to have rapid effect on reducing rectal temperature in mice and is a potent agonist of the G protein-coupled trace amine receptor TAR1 (Scanlan, 2009). A single dose of T 1 AM administered to rodents induces a hypometabolic state that in certain ways resembles hibernation. Monoam may be derived from low iodinated iodothyronines by aromatic amino acid decarboxylase ( Fig. 1 & 2), however, its role in developing mammals is not known.
Fig. 2. Postulated T 1 AM (monoam) formation. T 4 AM is not a substrate for type I or II deiodinase (D1 or D2) and cannot be deiodinated to T 3 AM. However, T 4 AM is readily deiodinated to rT 3 AM by D3, and rT 3 AM can be further deiodinated to ultimately provide T 1 AM. This suggests a unique biosynthetic deiodination pathway for T 1 AM starting from the decarboxylation products of either T 4 or rT 3 .

Sulfoconjugation of Iodothyronines
Sulfoconjuation has been found to be a major pathway for thyroid hormone metabolism in mammalian fetuses ( Fig. 1; Burrow et al., 1994;Wu et al., 2005;Simpson et al., 2005). The sulfation of thyroxine (T 4) and triiodothyronine (T 3 ) and their metabolites [reverse T 3 (rT 3 ) and 3,3'-diiodothyronine (T 2 )] may accelerate their further degradation and excretion. The sulfation of T 4 completely blocks the outer-ring deiodination to T 3 S. In addition, sulfated www.intechopen.com iodothyronines may serve as a reservoir for biologically active hormones such as T 3 , which can be recovered from T 3 S by sulfatases in selective tissues .
By far, T 2 is the preferred substrate for various mammalian sulfotransferases (SULTs). The purpose of rapid sulfation of T 2, as well as rT 3 in some tissues studied, is unknown. It is interesting that T 2 has been found to stimulate mitochondrial respiration in various rat tissues (Moreno et al., 1997) and rT 3 may play a role in regulating actin polymerization in brain cells (Leonard & Farwell, 1997). Also, sulfated T 2 is the preferred substrate for both human and rat arylsulfatase (ARS) in the microsomal fraction of liver and placenta . Thus, the possibility that these T 4 metabolites may play a physiological role in developing animals cannot be excluded.

Sulfoconjugation of Iodothyronines and their placental transfer in sheep
Before the onset of active synthesis and release of TH, iodothyronines detected in the fetus clearly are maternal in origin. This period is approximately the first 17 gestational days (d) in rats, 50d in sheep (Fig. 3) and 90d in humans. The proposed scheme for ovine fetal iodothyronine metabolism in late gestation (near term) depicts the production rates for sulfoconjugated thyroid hormone analogs (shown as numbers in parentheses along the thick arrows in Fig. 3). The high production rate (ug/kg/d) of T 4 sulfate (T 4 S) reflects the activity of the sulfation pathway. The rT 3 S production rate likely represents both sulfation of rT 3 and inner-ring deiodination of T 4 S . This scheme shown in Fig. 3 also predicts 3,3'-T 2 S is a major thyroid hormone metabolite in the fetus.
The transfer of TH and their metabolites may be a two-way street. We have shown high concentrations of sulfated iodothyronine analogs in human and ovine fetal serum. These include T 4 S, T 3 S, rT 3 S, and 3,3'-T 2 S (T 2 S) . The high gradient between fetal and maternal serum concentrations of iodothyronine sulfates raises the possibility that there may be significant fetal to maternal transfer of iodothyronine sulfoconjugates. When the ovine fetus was infused with pharmacological amounts (0.46 µmoles) of T 3 or T 3 S, significant fetal to maternal transfer of T 2 S and T 3 S occurred (Wu et al., 1995;Wu et al., 1999;Wu et al., 2006). It is noteworthy that significantly more T 3 S than T 2 S of fetal origin was recovered in maternal urine following the fetal infusion of either T 3 or T 3 S.
Furthermore, maternal T 2 S and T 3 S levels were significantly higher after fetal T 3 than after T 3 S infusion despite the fact that the mean fetal serum concentration of T 3 S after fetal T 3 S infusion was 20 times higher than following T 3 infusion (Wu et al., 1995). On the other hand, after fetal infusion of 125 I-T 3 , without disturbing the fetal stable T 3 pool, a mean of 19% of infused radioactive dose was recovered in maternal urine in 4 h. T 2 S, not T 3 S, was identified as the major radioactive iodothyronine in fetal to maternal transfer; only minimal amounts of T 3 S or T 3 , were found (Wu et al., 2006).
We also assessed the contribution of fetal TH to the urinary T 2 S and T 3 S pool in ewes. Maternal urinary T 2 S excretion (pmol/g creatinine) is significantly reduced by fetal thyroidectomy (Tx) but not by maternal Tx (Wu et al., 2001). Maternal urinary T 2 S excretion correlated positively with fetal serum T 4 concentrations but not with maternal serum T 3 or T 4 levels (Wu et al., 2001). In view of a possible functional role of T 2 to stimulate mitochondrial respiration, the removal of T 2 from fetal compartment may be necessary for normal maturation of mammalian fetuses . Furthermore, recent study showed that ARSC, the only sulfatase that hydrolyzes iodothyronine sulfates, has a substrate preference for T 2 (Kester et al., 2002), which raises the possibility that T 2 S could be readily reversed back to its precursor, an active iodothyronine. This would suggest a need for the fetus to remove T 2 S from fetal compartment. Furthermore, in the sheep model, T 2 S of fetal origin contributes significantly to maternal urinary T 2 S excretion and may reflect fetal iodothyronine production.

The Evaluation a T 2 S-crossreactive material (compound w) as a potential marker for fetal thyroid function
In humans, we found high levels of radioimmunoassayable T 2 S in maternal serum (Wu et al., 1994) and urine (Wu et al., 1998). Levels increased with the progression of pregnancy and peaked before parturition. At delivery, a 20-fold increase in serum "T 2 S" was found compared to non-pregnant women and "T 2 S" levels returned to non-pregnant values in 7 to 10 days (Fig. 4 & 5). On closer examination, the radioimmunoassayable "T 2 S" did not cochromatograph with synthetic T 2 S by HPLC (Wu et al., 1994). The authentic T 2 S was www.intechopen.com hydrolyzed by hot-acid digestion (Wu et al., 1994). Using this procedure, the recovery of T 2 S-crossreactive material was near 82% in fetal and maternal serum ( Fig. 6; Wu et al., 2007).  www.intechopen.com Fig. 6. Recovery of T 2 S-crossreactive material following hot-acid treatment. Top: Percent of T 2 S-crossreactive fraction that is hot-acid stable in cord (n = 6) and maternal (n = 12) serum samples.
The maternal and paired cord serum concentrations of T 2 S-reactive material presented were adjusted by this percentage to obtain the hot-acid-resistant T 2 S-equivalent activity, or the "corrected" value. Over 40 known synthetic thyroid hormone analogs were examined and none was found to be identical to the T 2 S-like material in pregnant women's serum. Thus, the name Compound W was given. It is postulated that W is a side-chain modification of T 2 S, which cross-reacts with T 2 S antibody but is slightly more hydrophobic than T 2 S. A possible candidate is N, N-dimethylated T 2 S ( Fig. 7; Wu et al., 2007).
To explore the possible origin of compound W, the serum concentrations of sulfated iodothyronines from cord arterial and venous samples were compared. There were no significant differences between the mean T 3 S, T 4 S, or rT 3 S concentrations of arterial and venous serum samples. However, the venous T 2 S-equivalent concentration was higher than arterial in seven of the paired samples and lower in two. The mean "corrected" W concentration in paired arterial/venous cord serum was found to be significantly higher in venous samples than in arterial samples (Wu et al., 2007). In addition, the mean of the maternal serum concentrations of T 2 S-reactive material was significantly lower than that of the paired cord serum concentrations. The rapid disappearance of W from maternal serum immediately after delivery supports this hypothesis (Wu et al., 1994). A similar disappearance slope of serum W was also found in newborn infants (Chen et al., 2010). These findings support the postulation that W is produced in the placenta.  Prior studies suggest strongly that compound W is a metabolite of fetal thyroid hormone capable of transplacental fetal to maternal transfer Wu et al., 1994;Cortelazzi et al., 1999;Wu et al., 2007). Both maternal and fetal compound W levels increase progressively during gestation with significant direct correlation (in both mothers and fetuses). Additionally, in 436 paired cord and maternal sera obtained at delivery, a highly significant positive correlation was observed between fetal and maternal compound W (Fig.  8). A significant positive correlation was also observed between serum levels of fetal compound W and fetal FT 4 and between maternal and fetal compound W ( Fig. 9; Cortelazzi Fig. 9. Compound W levels in fetal serum: correlation with serum fetal FT4 (n=29) and maternal compound W (n=42). [Cortelazzi et al., 1999, reproduced , 1999) whereas no correlation was observed between maternal serum compound W and maternal serum FT 4 in euthyroid or hyperthyroid women. Furthermore, maternal compound W levels seem to reflect the effects of drugs on fetal thyroid function (Cortelazzi et al., 1999). In women on propylthiouracil (PTU), maternal compound W levels were in the low normal range and did not show the usual increase with progression of gestation (Cortelazzi et al., 1999). The lack of progression in maternal compound W levels was confirmed in a recent study of 22 pregnant women treated with anti-thyroid medication ( Fig. 10; Vanmiddlesworth et al., 2011). A significant increase in maternal compound W was observed when the PTU dose was decreased or discontinued. Fig. 10. Courses of compound W in serum of pregnant patients with hyperthyroidism receiving antithyroid therapy. Two or more points on dotted lines mark the course of compound W in each patient, and the thick continuous black line shows the marginal mean course. Thick gray lines represent the 10 th (lower) and 90 th (upper) percentiles. Square markers on solid lines indicate suppressed or nonprogressive serial measurements in 5 patients; triangular markers on solid lines denote 2 patients who had transient progression and then reduction below the 10 th percentile at term.
Cortelazzi, et al. (Cortelazzi et al., 1999) suggest that abnormal thyroid function will be revealed by an absence of the normal rise of compound W during gestation. In comparison with relatively low incidence of congenital hypothyroidism in this population (Rastogi & LaFranchi, 2010;Harris & Pass, 2007), serial measurements of compound W in maternal serum can be considered a safe and practical test for the assessment of fetal thyroid function, particularly in hyperthyroid women treated with anti-thyroid drugs, whose fetuses can become hypothyroid due to the transplacental passage of the drug ( Fig.  10; Vanmiddlesworth et al., 2011).
Further study is needed to include compound W levels in a greater number of hyperthyroid pregnant women treated with anti-thyroid medication or pregnant women with evidence of autoimmune thyroid illnesses, followed by long-term follow-up of psychomotor development in these seemingly euthyroid babies. Suppressed W levels may help to define the sub-group of children exposed to PTU in utero or born to mothers with autoimmune disease who have been shown to have an adverse cognitive outcome (Grasberger & Refetoff, 2011;Rovet et al., 1999;Mirabella et al., 2000;Mitchell & Klein, 2004;Casey et al., 2005).

Conclusion
Sulfoconjugation is a major metabolic pathway for thyroid hormone in developing mammals. The significant rise of sulfated iodothyronines in mammalian fetal compartments raises the possibility that significant fetal to maternal transfer of the conjugates may occur in late gestation as the fetal hypothalamic-pituitary-thyroid system becomes more mature. This transfer may be a novel mechanism to maintain low T 3 states or regulate serum T 2 , a thermogenic hormone that is important for normal tissue maturity. The possibility that the transferred iodothyronine sulfate, especially T 2 S and its metabolite, may serve as a marker of fetal thyroid function needs to be further explored.