T3 Sulfate Research Articles

Serum iodothyronine concentrations in intestinally decontaminated rats treated with a 5′-deiodinase type I inhibitor 6-anilino-2-thiouracil

Enteric bacteria have been postulated to have a role in thyroid economy by promoting the hydrolysis of thyroid hormone conjugates of biliary origin, thus permitting the absorption and recycling of thyroxine (T4) and triiodothyronine (T3). An enterohepatic circulation of T3 might be more pronounced under conditions in which type I iodothyronine deiodinase activity (5'D-I) is inhibited, because this augments the accumulation of T3 sulfate conjugates in bile. This potential of increased gut reabsorption of T3 might explain, at least in part, the failure of serum T3 values to decrease appreciably when marked reductions in peripheral 5'D-I activity are induced by selenium deficiency or 6-anilino-2-thiouracil (ATU) administration. Thus, studies were performed to determine the effect of intestinal decontamination, in the absence and in the presence of 5'D-I inhibition, on plasma T4 and T3 concentrations. Groups of adult male rats received either enteric antibiotics or no antibiotics for 12 days and then, in half of the rats in each group, treatment for 10 days with ATU, a 5'D-I inhibitor that does not affect thyroid hormone synthesis. The activity of intestinal arylsulfatase and arylsulfotransferase, enzymes that catalyze hydrolysis of thyroid hormone conjugates, was reduced markedly by approximately 87% in rats that received antibiotics, regardless of whether or not they also received ATU. The ATU treatment markedly inhibited liver 5'D-I activity in antibiotic-treated as well as in non-antibiotic-treated rats (control = 399 +/- 32 U/mg protein (mean +/- SEM); ATU = 152 +/- 17: antibiotics = 351 +/- 29; antibiotics + ATU = 130 +/- 10; p < 0.01) and significantly increased plasma T4 and T3 sulfate (T4S, T3S) concentrations (control: T4S = 2.8 +/- 0.4 and T3S = 6.7 +/- 1.3 ng/dl; ATU: T4S = 6.2 +/- 1.4 and T3S = 10.6 +/- 2.1 ng/dl; antibiotics: T4S = 1.8 +/- 0.2 and T3S = 3.6 +/- 1.0 ng/dl; antibiotics + ATU: T4S = 6.8 +/- 0.7 and T3S = 9.7 +/- 1.8 ng/dl; p < 0.05). The ATU treatment was associated with a significant increase in plasma T4 and rT3 concentrations but did not affect plasma T3 concentrations, and intestinal decontamination did not alter these ATU-associated effects on circulating thyroid hormones. These results suggest that anaerobic enteric bacteria in the rat do not have an important role in recycling of thyroid hormones, either under normal conditions or in circumstances where 5'D-I activity is markedly reduced, and that increased gut absorption of T3 from T3S cannot explain the near-normal serum T3 values found when peripheral 5'D-I activity is markedly decreased.

Study of serum 3,5,3′-triiodothyronine sulfate concentration in patients with systemic non-thyroidal illness

Sulfation is an important pathway of triiodothyronine (T3) metabolism. Increased serum T3 sulfate (T3S) values have been observed during fetal life and in pathological conditions such as hyperthyroidism and selenium deficiency. Similar variations have also been reported in a small number of patients with systemic non-thyroidal illness, but the underlying mechanisms have not been elucidated. In this study, serum T3S concentrations have been measured by a specific radioimmunoassay in 28 patients with end-stage neoplastic disease (ESND) and in 44 patients with chronic renal failure (CRF); 41 normal subjects served as controls. Both ESND and CRF patients had lower serum total T4 (TT4) and total T3 (TT3) than normal controls, while serum reverse T3 (rT3) was increased significantly in ESND (0.7 +/- 0.5 nmol/l; p < 0.001 vs. controls) but not in CRF (0.3 +/- 0.1 nmol/l). The TT3/rT3 ratio, an index of type I iodothyronine monodeiodinase (type I MD) activity, was reduced significantly in both groups of patients. Serum T4-binding globulin (TBG) was decreased in CRF but not in ESND patients. Serum T3S was significantly higher both in ESND (71 +/- 32 pmol/l) and CRF (100 +/- 24 pmol/l) than in controls (50 +/- 16 pmol/l, p < 0.001). Serum T3S values showed a positive correlation with rT3 values and a negative correlation with both TT3 and FT3 values in ESND, but not in CRF. In the latter group a positive correlation was observed between T3S and TBG values. The T3S/FT3 ratio was higher both in CRF (18 +/- 5) and in ESND (23 +/- 18) as compared to controls (10 +/- 4). Serum inorganic sulfate was increased and correlated positively with T3S values in CRF patients. In conclusion, the results of this study in a large series of patients confirm that patients with systemic non-thyroidal illness have increased serum T3S levels. The mechanisms responsible for these changes appear to be different in ESND and CRF patients. In ESND the increase in serum T3S levels is mainly related to reduced degradation of the hormone by type I MD, whereas in CRF it might be driven by the enhanced sulfate ion concentration, and could be partially dependent on the impaired renal excretion of T3S. Because T3S can be reconverted to T3, it is possible that increased T3S concentrations contribute to maintenance of the euthyroid state in systemic non-thyroidal disease.

Human Liver Triiodothyronine Sulfotransferase: Copurification with Phenol Sulfotransferases

To ascertain whether triiodothyronine (T3) sulfotransferase coeluted with the known phenol sulfotransferases (PSTs) during purification, human liver thermostable PST, thermolabile PST, and T3 sulfotransferase were assayed with p-nitrophenol, dopamine, and T3, respectively. Thermostable PST eluted from an ion-exchange column in two sequential peaks of activity (Peaks I and II), followed by a peak of thermolabile PST activity. There were three peaks of T3 sulfotransferase with thermostable PST: two within thermostable PST Peak I, and one peak of T3 sulfotransferase activity within thermostable PST Peak II. There was a minor peak of T3 sulfotransferase with thermolabile PST. Further purification of thermostable Peak I showed coelution of T3 sulfotransferase with thermostable PST during gel filtration and affinity chromatography. SDS-PAGE revealed a major protein band at 31 kDa. Dehydroepiandrosterone sulfotransferase comprised only 4% of the final activity. This report demonstrates coelution of T3 sulfotransferase with thermostable PST, shows a potential additional isozyme of T3 sulfotransferase, and points out the apparent minimal role of dehydroepiandrosterone sulfotransferase in T3 sulfation. The findings support the hypothesis that thermostable PST is the predominant human liver T3 sulfotransferase activity.

Identification of 3,3'-T2S as a fetal thyroid hormone derivative in maternal urine in sheep

We measured 3,3'-diiodothyronine sulfate (T2S) in serum and urine (n = 5-6) obtained from euthyroid fetal (94-145 days of gestation, term = 150 days), newborn, and adult sheep and in serum and urine samples from ovine fetuses 13 days after total thyroidectomy conducted between 110 and 113 gestation days (n = 5). Sham-operated twin fetuses served as controls (n = 5). Mean serum T2S concentrations increased progressively from 94 days (74 ng/dl) to 130 days (420 ng/dl), decreasing thereafter to 145 days (197 ng/dl). T2S concentrations in fetal urine peaked at 110 days (117 ng/dl). In hypothyroid fetuses, mean serum and urine T2S were 60 and 53% of control values. To assess the possibility that the T2S in maternal serum/urine is derived from fetal serum 3,5,3'-triiodothyronine (T3), we measured T3, T3 sulfate (T3S), and T2S in fetal serum and in maternal serum and urine after bolus infusion of T3 to the fetus (n = 4). Additionally, T3, T3S, and T2S concentrations were measured in maternal serum and urine after T3 infusion to the maternal ewes (n = 4). Fetal T3 infusion rapidly increased fetal serum T3S and T2S. Maternal serum and urine T3S and T2S concentrations increased, whereas T3 concentrations remained unchanged. Maternal T3 infusion increased in serum and urine T3S and T2S levels, but the levels, relative to T3, were less than values measured after fetal T3 infusion. We conclude that T2S is a normal thyroid hormone metabolite in the ovine fetus and suggest that a major pathway of fetal T2S production is T3 to T3S to T2S.(ABSTRACT TRUNCATED AT 250 WORDS)

Sulfation after deiodination of 3,5,3'-triiodothyronine in rat cultured astrocytes.

The metabolism of [125I]T3 by rat astrocytes in culture was analyzed by Sephadex LH-20 chromatography and HPLC. The conjugates isolated on LH-20 were not hydrolyzed by glucuronidase, indicating the absence of glucuroconjugates. 3,3'-Diiodothyronine (3,3'T2) sulfate (3,3'T2-S) was the main product that accumulated in the medium over the T3 concentration explored (10 pM to 10 nM). The identity of the peak eluted as 3,3'T2-S was ascertained by its hydrolysis with sulfatase and the generation of 3,3'T2 identified by HPLC. 3'-Monoiodothyronine sulfate was also found in cells treated with 1 microM retinoic acid, i.e. with high type III deiodinase activity. No T3 sulfate (T3-S) was found as a metabolite of T3. Astrocytes did not break down 1 nM [125I]T3-S added to the medium. Astrocytes pretreated for 3 days with 10 nM T3 showed increased production of 3,3'T2-S from 10 nM [125I]T3. Exogenous [125I]3,3'T2 (20 nM) was conjugated to 3,3'T2-S released into the medium. Pretreatment of astrocytes with 10 nM T3 did not alter the production of 3,3'T2-S from 3,3'T2. Thus, T3 is metabolized in astrocytes by direct 5-deiodination, followed by sulfation. Whereas T3 induces its own deiodination and type III deiodinase activity, T3 does not regulate the sulfation of its main metabolite, 3,3'T2. This demonstration of sulfation of iodothyronines in cells originating from the brain raises the question of the role of this TH metabolic pathway in the brain.

Sulfation after deiodination of 3,5,3'-triiodothyronine in rat cultured astrocytes

The metabolism of [125I]T3 by rat astrocytes in culture was analyzed by Sephadex LH-20 chromatography and HPLC. The conjugates isolated on LH-20 were not hydrolyzed by glucuronidase, indicating the absence of glucuroconjugates. 3,3'-Diiodothyronine (3,3'T2) sulfate (3,3'T2-S) was the main product that accumulated in the medium over the T3 concentration explored (10 pM to 10 nM). The identity of the peak eluted as 3,3'T2-S was ascertained by its hydrolysis with sulfatase and the generation of 3,3'T2 identified by HPLC. 3'-Monoiodothyronine sulfate was also found in cells treated with 1 microM retinoic acid, i.e. with high type III deiodinase activity. No T3 sulfate (T3-S) was found as a metabolite of T3. Astrocytes did not break down 1 nM [125I]T3-S added to the medium. Astrocytes pretreated for 3 days with 10 nM T3 showed increased production of 3,3'T2-S from 10 nM [125I]T3. Exogenous [125I]3,3'T2 (20 nM) was conjugated to 3,3'T2-S released into the medium. Pretreatment of astrocytes with 10 nM T3 did not alter the production of 3,3'T2-S from 3,3'T2. Thus, T3 is metabolized in astrocytes by direct 5-deiodination, followed by sulfation. Whereas T3 induces its own deiodination and type III deiodinase activity, T3 does not regulate the sulfation of its main metabolite, 3,3'T2. This demonstration of sulfation of iodothyronines in cells originating from the brain raises the question of the role of this TH metabolic pathway in the brain.

Metabolism of sulfoconjugated thyroid hormone derivatives in developing sheep

Although the production of thyroxine (T4) in the developing ovine fetus ranges from 20 to 50 micrograms.kg-1.day-1, production rates for 3,5,3'-triiodothyronine (T3) average only 1-2 micrograms.kg-1.day-1, whereas reverse T3 (rT3) production rates approach 5-6 micrograms.kg-1.day-1. Thus the fate of the majority of fetal T4 production is uncertain. Recently we have reported significant concentrations of various thyroid hormone sulfoconjugates in serum and other fetal compartments. In the present study, we used steady-state kinetic techniques in developing sheep to establish the clearance and production rates for T4, T3, and rT3 sulfates. These studies confirm that T4, T3, and rT3 sulfate are predominant metabolites of thyroid hormone in the developing ovine fetus. Plasma clearance rates for T3, T4, and rT3 sulfates are low in the fetus, averaging 0.67 +/- 0.07, 1.46 +/- 0.11, and 4.1 +/- 1 ml.kg-1.min-1, respectively. Clearance rates for these thyrosulfoconjugates increase two to fourfold postnatally, probably reflecting increased activity of 5'-monodeiodinase after birth. Moreover, fetal production rates for these sulfated thyroid hormone metabolites exceed those of 2-wk-old sheep 4- to 10-fold. The data suggest that a significant route of fetal T4 metabolism is sulfation followed by deiodination to rT3 sulfate.

Activation and inactivation of thyroid hormone by type I iodothyronine deiodinase

The prohormone thyroxine (T4) is activated by outer ring deiodination (ORD) to 3,3′,5-triiodothyronine (T3) and both hormones are degraded by inner ring deiodination (IRD) to 3,3′,5′-triiodothyronine (rT3) and 3,3′-diiodothyronine, respectively. Indirect evidence suggests that the type I iodothyronine deiodinase (ID-I) in liver has both ORD and IRD activities, with preference for rT3 and sulfated iodothyronines as substrates. To establish this, we have compared the ORD of rT3 and IRD of T3 and T3 sulfate by homogenates of cells transfected with rat ID-I cDNA and by rat liver microsomes. In both preparations rT3 is the preferred substrate, while deiodination of T3 is markedly accelerated by its sulfation. Kinetic analysis provided similar K m and V max values in cell homogenates and liver microsomes. These data demonstrate unequivocally that ID-I is capable of both activating and inactivating thyroid hormone by ORD and IRD, respectively.

3,5,3'-Triiodothyronine (T3) sulfate: a major metabolite in T3 metabolism in man.

Previous studies have suggested that T3 metabolism relies more on nondeiodinative conjugation than on direct deiodinative degradation for its disposal in man. To better define this process, tracer T3 kinetic studies were performed in five euthyroid subjects before and after iopanoic acid (IA) administration to selectively impair T3 deiodinative disposal. Both a low IA (0.5-g load, followed by 0.5 g/day for 7 days) and a high IA (3.0-g load, followed by 3.0 g/day for 7 days) dosing schedule were employed to achieve varying levels of deiodinase inhibition. Additionally, the high IA dose was repeated with simultaneous oral T3 administration (100 micrograms daily) to normalize serum T3 levels that were reduced by IA-induced inhibition of T4 to T3 conversion. The results demonstrated that baseline serum T3 (2.3 +/- 0.1 nmol/L) and T3/T4 (1.9 +/- 0.1 x 10(-2)) values were significantly reduced by both the low IA (1.5 +/- 0.1 nmol/L and 1.2 +/- 0.1 x 10(-2), respectively) and the high IA (1.5 +/- 0.1 nmol/L and 0.9 +/- 0.2 x 10(-2), respectively) dosing schedule and that the addition of oral T3 to the high IA regimen restored both the T3 and T3/T4 levels to near-normal values (2.9 +/- 0.3 nmol/L and 1.7 +/- 0.2 x 10(-2), respectively). Low IA also significantly decreased T3 clearance (30 +/- 4 to 18 +/- 2 L/day; P < 0.005) and fractional urinary tracer recovery (70 +/- 3% to 37 +/- 4%; P < 0.005), whereas high IA produced only a minimal further reduction in clearance (16 +/- 2 L/day; P < 0.01) and urinary tracer recovery (32 +/- 3%; P < 0.05). Surprisingly, oral administration of T3 to the high IA regimen significantly increased T3 clearance (23 +/- 4 L/day; P < 0.01) without changing urinary tracer recovery (34 +/- 5%) compared to the effects of high IA alone. Evaluation of the urinary T3 metabolite pattern demonstrated that the major products of T3 metabolism were T3 sulfate and 3,3-diiodothyronine sulfate. These observations confirm previous results suggesting that the majority of nondeiodinative T3 disposal occurs via T3 sulfate formation. The additional finding that such nondeiodinative disposal may also be influenced by the circulating T3 level leads us to propose that sulfotransferase enzyme systems may play an important role in regulating the prereceptor availability of this ligand.

A study of the 3,5,3'-triiodothyronine sulfation activity in the adult and the fetal rat.

We have employed a new in vitro assay for study of the T3 sulfation activity in rat tissues. The assay measures by RIA the generation of T3 sulfate (T3S) during incubation of T3 with cytosol of rat tissues as the source of phenol sulfotransferase(s) and 3-phosphoadenosine-5'-phosphosulfate as the sulfate donor. The conversion of T3 to T3S proceeded rapidly for 30 min at 37 C, and the optimal pH of the reaction was 8.0. Heating the cytosol at 44 C for 15 min decreased T3S production to 63% of its value at 37 C. T3 sulfation activity was plentiful in rat liver, brain, and kidney, but little activity was demonstrable in other tissues. The Km and maximum velocity of the hepatic conversion of T3 to T3S were 114 microM and 159 pmol/mg protein.h, respectively. There was a marked inhibition of the conversion of T3 to T3S with salicylamide, 3'-monoiodothyronine, thyronine, and rT3; the IC50 of these inhibitors approximated 15, less than 0.1, 9.5, and 43 microM, respectively. On day 17 of gestation, the T3 to T3S conversion activity was more abundant in fetal skin than in other fetal tissues. However, the activity decreased in fetal skin while it increased in fetal liver, kidney, and brain nearer to term on day 20. Placenta demonstrated lower T3 to T3S conversion activity than several fetal or maternal tissues. There was no effect of hypothyroidism or hyperthyroidism on T3 sulfation activity. We conclude that T3 sulfation activity in the rat is 1) most abundant in liver, kidney, and brain tissues of the adult; 2) inhibited more avidly by 3'-monoiodothyronine than other thyronines; 3) very abundant in fetal skin early in gestation; and 4) little affected by the thyroidal status of the animal.

A study of the 3,5,3'-triiodothyronine sulfation activity in the adult and the fetal rat

We have employed a new in vitro assay for study of the T3 sulfation activity in rat tissues. The assay measures by RIA the generation of T3 sulfate (T3S) during incubation of T3 with cytosol of rat tissues as the source of phenol sulfotransferase(s) and 3-phosphoadenosine-5'-phosphosulfate as the sulfate donor. The conversion of T3 to T3S proceeded rapidly for 30 min at 37 C, and the optimal pH of the reaction was 8.0. Heating the cytosol at 44 C for 15 min decreased T3S production to 63% of its value at 37 C. T3 sulfation activity was plentiful in rat liver, brain, and kidney, but little activity was demonstrable in other tissues. The Km and maximum velocity of the hepatic conversion of T3 to T3S were 114 microM and 159 pmol/mg protein.h, respectively. There was a marked inhibition of the conversion of T3 to T3S with salicylamide, 3'-monoiodothyronine, thyronine, and rT3; the IC50 of these inhibitors approximated 15, less than 0.1, 9.5, and 43 microM, respectively. On day 17 of gestation, the T3 to T3S conversion activity was more abundant in fetal skin than in other fetal tissues. However, the activity decreased in fetal skin while it increased in fetal liver, kidney, and brain nearer to term on day 20. Placenta demonstrated lower T3 to T3S conversion activity than several fetal or maternal tissues. There was no effect of hypothyroidism or hyperthyroidism on T3 sulfation activity. We conclude that T3 sulfation activity in the rat is 1) most abundant in liver, kidney, and brain tissues of the adult; 2) inhibited more avidly by 3'-monoiodothyronine than other thyronines; 3) very abundant in fetal skin early in gestation; and 4) little affected by the thyroidal status of the animal.

Thyromimetic effects of 3,5,3'-triiodothyronine sulfate in hypothyroid rats

Several parameters of the effects of thyroid hormone were examined in hypothyroid thyroidectomized (Tx) rats treated with T3 sulfate (T3S) or T3 [0.46 (low dose) or 2.3 (high dose) nmol/day for 10 days, ip]. Tx rats showed a marked degree of growth retardation, which improved significantly after treatment with both doses of T3S and T3. The mean serum GH level was markedly reduced in Tx rats, and it improved significantly to similar levels after treatment with the high dose of T3S and the low dose of T3. Type I monodeiodinase (MD) activity was markedly reduced in liver and kidney tissues of Tx rats. It increased significantly in Tx rats treated with the high dose of T3S; the latter values were similar to those observed in Tx rats treated with the low dose of T3. Hepatic and renal type I MD activities increased to supranormal levels in Tx rats treated with the high dose of T3. Cardiac outer ring (5') monodeiodination of 3',5'-diiodothyronine to 3'-monoiodothyronine was also significantly reduced in Tx rats, but it improved significantly only after treatment with the high dose of T3. Type III 5-MD activity was significantly reduced in the cerebral cortex of Tx rats. It was restored to normal in Tx rats treated with the high dose of T3S and both doses of T3. Serum TSH, markedly elevated in Tx rats, was appreciably reduced only in rats treated with the high dose of T3. In another study, significant suppression of serum TSH was observed when Tx rats were treated with T3S (11.5 nmol/day) or T3 (2.3 nmol/day) for 3 days. We conclude that administration of T3S to hypothyroid rats produces thyromimetic effects, with a potency approximately one fifth that of T3.

A study of the serum 3,5,3'-triiodothyronine sulfate concentration in normal and hypothyroid fetuses at various gestational stages.

We have studied T3 sulfate (T3S) levels, blindly, in coded plasma samples from 21 normal and 3 hypothyroid fetuses at different stages of gestation (19-42 weeks). Fetal plasma samples were obtained by cordocentesis. T3S was detectable in all samples studied, with values ranging from 50-294 (mean +/- SD, 130 +/- 62 pmol/L). Plasma T3S was low (< 45 pmol/L) in all 4 normal adult control subjects studied simultaneously; serum T3S ranged from less than 20 to 130 in another set of 18 control subjects (mean +/- SD, 63 +/- 32 pmol/L). Fetal T3S values were positively correlated with gestational age (r = 0.43; P < 0.05), but not with free T4 (FT4), FT3, or TSH values. In the 3 hypothyroid fetuses at 31, 38, and 40 weeks gestation, respectively, plasma TSH was elevated (26, 98, and 24 mU/L, respectively), FT4 was low (10, 6.7, and 7.5 pmol/L, respectively), and FT3 was normal or high (3.2, 8.2, and 2.2 pmol/L, respectively). However, T3S values in hypothyroid fetuses (88, 133, and 252 pmol/L, respectively) were similar to those in normal fetuses at corresponding gestational ages. We conclude that 1) T3S is detectable in fetal circulation from at least 19 weeks gestation, and its concentration increases with fetal-age; 2) plasma T3S concentrations in the fetus at 19-40 weeks gestation are at least comparable to but generally higher than those in the adult; and 3) plasma T3S levels in hypothyroid fetuses are similar to those in normal fetuses. Recent studies demonstrating the ability of some fetal rat tissues (e.g. cerebral cortex) to desulfate T3S to T3 have suggested a possible role of T3S as a source of T3. Normal T3S in fetal hypothyroidism suggests that T3S may contribute to attenuation of the effects of hypothyroidism during intrauterine life.

The development of a radioimmunoassay for reverse triiodothyronine sulfate in human serum and amniotic fluid.

Sulfated iodothyronines including T4-sulfate (T4S) and T3-sulfate (T3S) have been identified in human serum and amniotic fluid. Little is known, however, about the existence of sulfate conjugation of reverse T3 (rT3S) in man. In this report, we employed a novel, sensitive, and specific rT3S RIA to address this question. The rabbit antiserum to rT3S was highly specific; T4, T3, rT3, and 3,3'-T2 showed less than 0.002% cross-reaction with the antiserum. Only T4S and T3S cross-reacted significantly (0.3% and 0.01%, respectively); other analogs cross-reacted less than 0.0001%. The detection threshold of the RIA was 14 pmol/L (1.0 ng/dL). The mean serum rT3S concentration (pmol/L) was 40 in euthyroid subjects. Values were similar in hypothyroid patients (38) and pregnant women (52) but significantly (P < 0.01) elevated to 176 in hyperthyroid patient, 74 in patients with nonthyroid illnesses, and 684 in cord sera of newborns. Serum rT3S increased significantly in hyperthyroid patients 1 day after administration of 1 g sodium ipodate orally. Reverse T3S was detected consistently in amniotic fluid at 14 to 22 weeks of gestation and showed a marked rise 1-3 weeks after intraamniotic administration of 500-1000 micrograms T4. The various data suggest that: (1) rT3S is a normal component of human serum and amniotic fluid; (2) it is derived from metabolism of T4 or rT3; (3) circulating rT3S increases in hyperthyroidism and in circumstances where type I 5'-monodeiodinating activity is low, e.g. nonthyroid illnesses, fetal life, and after administration of ipodate.

A radioimmunoassay for measurement of thyroxine sulfate.

A highly sensitive, specific, and reproducible RIA has been developed to measure T4 sulfate (T4S) in ethanol extracts of serum. rT3 sulfate (rT3S) cross-reacted 7.1%, and T3S cross-reacted 0.59% in the RIA; T4, T3, rT3, and 3,3'-diiodothyronine cross-reacted 0.004% or less. The recovery of nonradioactive T4S added to serum averaged 95%. The detection threshold of the RIA was 18 pmol/L. The coefficient of variation averaged 6.9% within an assay and 12% between assays. T4S was bound by T4-binding globulin and albumin in serum. The free fraction of T4S in four normal sera averaged 0.06% compared to a value of 0.03% for T4 (P < 0.001). The serum concentration of T4S was (mean +/- SE) 19 +/- 1.2 pmol/L in normal subjects, 33 +/- 10 in hyperthyroid patients with Graves' disease, 42 +/- 15 in hypothyroid patients, 34 +/- 6.9 in patients with systemic nonthyroidal illnesses, 21 +/- 4.3 in pregnant women at 15-40 weeks gestation, and 245 +/- 26 in cord blood sera of newborns; the value in the newborn was significantly different from normal (P < 0.001). The mean concentration of T4S in amniotic fluid samples at 15-38 weeks gestation was 106 +/- 22 pmol/L (cf. normal adults; P < 0.001). Administration of sodium ipodate (Oragrafin; 3 g, orally) to hyperthyroid patients was associated with a transient increase in serum T4S. The T4S content of the thyroid gland was less than 1/4000th that of T4. We conclude that 1) T4S is a normal component of human serum, and its levels are markedly increased in newborn serum and amniotic fluid; and 2) the sulfation pathway plays an important role in the metabolism of T4 in man.

Thyroxine sulfate is a major thyroid hormone metabolite and a potential intermediate in the monodeiodination pathways in fetal sheep.

T3 and rT3 production rates in the fetus account for roughly only a third of the total T4 production rate; thus, the fate of the majority of T4 produced in the fetus is unknown (the "T4 disposal gap"). We developed sensitive and specific T4 sulfate (T4S) and T3 sulfate (T3S) RIAs to investigate the roles of these compounds in fetal T4 metabolism. T3, T4, T3S, and T4S were determined in a variety of tissue fluid and/or serum samples obtained from fetal, newborn (n = 6), and adult (n = 6) sheep. Four groups of fetal animals, with gestational ages of 94 days (n = 5), 110-111 days (n = 6), 130-131 days (n = 6), and 145 days (n = 6; term = 150 days), were studied. In addition, type I 5'-monodeiodinase (5'-MDI) activity was quantified in liver and kidney tissues. 5'-MDI activities were lower in 94- to 131-day-old fetuses than in fetuses near term or in newborn animals. Mean serum T3 concentrations increased progressively from 94 days (19 ng/dl) to term (371 ng/dl), while mean T3S and T4S serum concentrations were highest at 130 days gestation (237 and 989 ng/dl), decreasing to term. Serum T3S and T4S concentrations decreased further in newborns and adult sheep. T4S and T3S levels in allantoic fluid were significantly higher than those in urine and amniotic fluid in all fetal age groups studied. T4S levels in bile were high from 94-130 days gestation (873-1006 ng/dl), decreasing by 50% at term (529 ng/dl). T4S concentrations in meconium were 46- to 83-fold higher than those in bile from 94 days gestation to term. In contrast, bile T3S levels increased progressively from 94-145 days gestation (191-605 ng/dl), while meconium T3S levels decreased during the same period (33-14 micrograms/100 g). These data demonstrate that 1) sulfated iodothyronines, particularly T4S, are major thyroid hormone metabolites in the fetus; 2) both T4S and T3S are excreted into bile and urine and concentrated in meconium and allantoic fluid; and 3) the high levels of T4S and T3S in serum and other fluids may reflect lower tissue type I 5'-MDI activities. We speculate that T4S and T3S may be further metabolized to other sulfated metabolites and may account in part for the T4 disposal gap in fetal sheep.

Effects of selenium deficiency on thyroid hormone economy in rats.

In selenium-deficient rats, peripheral T4 to T3 conversion is markedly decreased due to the loss of the selenoprotein, type I iodothyronine 5'-deiodinase (5'D-I). Despite the marked increase in circulating T4 that results from this loss of 5'D-I, serum T3 concentrations in selenium-deficient rats remain in the normal range. To determine the physiological mechanism(s) that maintains circulating T3 when peripheral T4 to T3 conversion is impaired, we examined the interrelationships between selenium intake and the metabolism of T3 and T4 in the rat. In euthyroid rats, selenium deficiency caused the expected loss of 5'D-I, with a 52% increase in serum T4, which paralleled an increase in the T4 biological half-life. Consistent with the prolonged t1/2 of T4, short term thyroidectomy (48 h) in selenium-deficient rats failed to decrease serum T4 concentrations to the levels observed in short term thyroidectomized, selenium-supplemented rats. Short term thyroidectomy also caused an expected 33% decrease in liver 5'D-I and a 44% increase in brain type II iodothyronine 5'-deiodinase (5'D-II) activities in selenium-supplemented rats. However, in selenium-deficient rats, short term thyroidectomy did not affect 5'D-I or 5'D-II activities. In contrast to the selenium-dependent changes in circulating T4 levels, little or no change in circulating T3 concentrations occurred. There was a 20% increase in the T3 half-life in selenium-deficient rats. The serum T3 sulfate concentration was increased, and T3 deiodination was reciprocally decreased in the selenium-deficient rats. These data suggest that increased T3 sulfate generation in selenium-deficient rats may lead to greater T3 availability through enterohepatic recycling of the iodothyronine and may explain why there are only minor changes in serum T3 concentrations in selenium-deficient rats.

A radioimmunoassay for measurement of 3,5,3'-triiodothyronine sulfate: studies in thyroidal and nonthyroidal diseases, pregnancy, and neonatal life.

A highly sensitive, specific, and reproducible RIA has been developed to measure T3 sulfate (T3S). Only T4 sulfate cross-reacted significantly (approximately 3%) in the RIA; rT3 sulfate, T4, T3, rT3, and diiodothyronine cross-reacted less than 0.01%. T3S was bound by thyronine-binding globulin and albumin in serum. The free fraction of T3S in four normal sera averaged 0.25% compared to a value of 0.35% for T3. Therefore, T3S was measured in ethanol extracts of serum. Recovery of the nonradioactive T3S added to serum averaged 92%. The dose-response curves of inhibition of binding of [125I]T3S to anti-T3S antibody by serial dilutions of serum extracts were essentially parallel to the standard curve. The detection threshold of the RIA was 20 pmol/L (1.5 ng/dL). The coefficient of variation averaged 7.8% within an assay and 11% between assays. The serum concentration of T3S was (mean +/- SE) 76 +/- 7.2 pmol/L in normal subjects, 268 +/- 29 in hyperthyroid patients with Graves' disease, 92 +/- 28 in hypothyroid patients, 201 +/- 32 in patients with systemic nonthyroidal illnesses, 40 +/- 6.2 in pregnant women (15-31 weeks gestation), and 429 +/- 39 in cord sera of newborns; the values in hyperthyroidism, nonthyroidal illnesses, and newborns were significantly different from normal (P less than 0.01). The mean concentration of T3S in amniotic fluid samples at 15-31 weeks gestation (90 +/- 1.3 pmol/L) was significantly higher than the corresponding value in maternal serum (P less than 0.05) and significantly lower than the corresponding value in newborn cord blood serum (P less than 0.001). Oral administration of sodium ipodate (Oragrafin; 3 g) to two hyperthyroid patients was associated with a 76-190% increase in serum T3S at 8 h, followed by a gradual decrease to a nadir that was 25-60% of the baseline value 2-3 days after ipodate ingestion. We conclude that 1) T3S is a normal component of human serum, and its levels change substantially in several physiological and pathological conditions; 2) sulfation pathway plays an important role in the metabolism of iodothyronines in man; and 3) high serum T3S levels in newborns and low normal levels in pregnancy despite elevated thyronine-binding globulin levels may signify markedly different metabolism of T3S in the mother and fetus.

Hepatic triiodothyronine sulfation and its regulation by growth hormone and triiodothyronine in rats.

The regulatory mechanism of cytosolic sulfation of T3 has been studied in rat liver. Sulfation of T3 is sexually differentiated in adult rats of Sprague-Dawley (SD), Fisher 344, and ACI strains. In SD strain, the male animals showed 4 times higher sulfating activity than did the females. The specific activity was decreased by hypophysectomy of male adult rats, but was not affected in the females. Thus, the sex-difference was abolished in the hypophysectomized condition. Supplement of human GH intermittently twice daily for 7 days, to mimic the male secretory pattern, increased T3 sulfating activity in both sexes of hypophysectomized rats, whereas continuous infusion to mimic a female secretory pattern had no appreciable effect. Cytosolic sulfation of T3 was decreased by 25 to 30% by thyroidectomy or propylthiouracil treatment of male adult rats, and was restored by the supplementation of T3 (50 micrograms/kg daily for 7 days) to thyroidectomized rats. Administration of T3 in hypophysectomized rats almost completely restored the sulfating activity in the males and increased the activity in the females. Cytosolic T3 sulfation was inhibited by the addition of known inhibitors of phenol sulfotransferase, pentachlorophenol or 2,6-dichloro-4-nitrophenol. These results indicate a role of pituitary GH in hepatic sulfation of thyroid hormones in rats. The data obtained also raise the possibility that GH may modify the effect of thyroid hormones on the pituitary by a feed-back mechanism through changing the level of a sex-dominant phenol sulfotransferase(s) in rat livers. T3 was also sulfated in hepatic cytosols of mouse, hamster, rabbit, dog, monkey, and human.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport of 3,5,3'-triiodothyronine into the perfused rat liver and subsequent metabolism are inhibited by fasting

The effects of 48-h fasting on transport of T3 and subsequent metabolism in the isolated perfused rat liver were investigated. Tracer T3 disappearance curves from the recirculating medium consisted of a fast component (FC) and a slow component (SC). Using a two-compartment model, both transport [expressed as the fractional transport rate constant from medium to liver (k21)] and disposal of T3 were calculated. After fasting, k21, total metabolism, and metabolism corrected for differences in mass transfer were diminished, pointing to both decreased transport and metabolism, presumably caused by depletion of liver ATP. Concerning transport, it was shown that only transport into the intracellular liver compartment and not transport to the extracellular liver compartment was decreased after fasting. As for metabolism, T3 glucuronidation was diminished; T3 sulfation and subsequent deiodination were not affected. All mentioned decreased parameters normalized after the addition of a combination of insulin, cortisol, and/or glucose to the medium, possibly by (partially) restoration of cellular energy stores.