Euthyroid hyperthyroxinemia due to a generalized 5'-deiodinase defect

We studied an 11-yr-old girl with asymptomatic hyperthyroxinemia, who remained euthyroid and healthy for 5 yr of follow-up. Besides having elevated serum T4 concentrations, her serum free T4 concentrations were consistently elevated, as measured by three different methods, including equilibrium dialysis and ultrafiltration. Serum total and free T3 concentrations were in the low normal range, and serum 3,5-diiodothyronine (3,5-T2) levels were low, suggesting reduced 5'-deiodination of both T4 and T3. Serum total and free rT3 and total and free 3', 5'-T2 concentrations were all markedly elevated, whereas serum total and free 3,3'-T2 were low, suggesting unaltered 5-deiodination of T4 to rT3 and of rT3 to 3',5'-T2 in combination with reduced 5'-deiodination of rT3 and 3',5'-T2. The girl had a small diffuse goiter, her serum TSH response to TRH was exaggerated, and thyroid radioiodine uptake was elevated, suggesting slightly increased TSH secretion and, consequently, increased thyroid secretion. Both T3 and T4 administration resulted in suppressed basal as well as TRH-stimulated serum TSH concentrations, and radioiodine uptake was suppressed during T3 administration. Our data suggest reduced activity of several (all?) peripheral 5'-deiodination pathways, including possibly also thyrotroph T4 5'-deiodination. Thus, this girl seems to have a previously unrecognized syndrome of generalized 5'-deiodinase deficiency.     

Simultaneous turnover studies of thyroxine, 3,5,3' and 3,3',5'-triiodothyronine, 3,5-, 3,3'-, and 3',5'- diiodothyronine, and 3'-monoiodothyronine in chronic renal failure

The present study evaluates the sequential extra-thyroidal monodeiodination of thyroid hormones through tri-, di-, and monoiodothyronines in chronic renal failure (CRF) in man. Simultaneous turnover studies of T4, T3, rT3, 3,5-diiodothyronine (3,5-T2), 3,3'-T2, 3',5'-T2, 3'5'-T2, and 3'-monoiodothyronine (3--T1) were conducted in six patients with CRF (creatinine clearance, 9-18 ml/min) using the single-injection, noncompartmental approach. Serum levels of T4, T3, and 3,5-T2 were reduced to two thirds of control levels (P less than 0.05), whereas serum rT3 and 3,3'-T2 levels were reduced to a minor degree. Serum 3'-5'-T1 was doubled (p less than 0.05). The MCRs of T4, rT3, and 3',5'-T2 were enhanced to 168%, 127%, and 187% of normal (P less than 0.05), respectively, whereas those of T3, 3,5-T2, 3,3'-T2, and 3'-T1 were unaffected. The mean production rates (PRs) of the iodothyronines in CRF were as follows (CRF vs. control values, expressed as nanomoles per day/70 kg): T4, 119 vs. 125; T3, 26 vs. 44 (P less than 0.01); rT3, 49 vs, 48; 3,5-T2, 3.5 vs. 7.2 (P less than 0.001); 3,3'-T2, 25 vs. 35 (P less than 0.01); 3',5'-T2, 25 vs. 14 (P less than 0.01); and 3'-T1, 39 vs. 30. Previous studies have demonstrated reduced phenolic ring (5'-) deiodination of T4 in CRF, which is supported by the present finding of unaltered PR of T4 and reduced PR of T3. In contrast the 5'-deiodination of T3 leading to the formation of 3,5-T2 was found unaffected by CRF, since the conversion rate (CR) of T3 to 3,5-T2 (PR 3,5-T2/PR T3) was unaltered (16% vs. 15% in controls). The tyrosylic ring (5-) deiodination of T4 to rT3 was unaffected in patients with CRF, the CR being 42% vs. 40% in controls, in contrast to an enhanced CR of rT3 to 3',5'-T2 (53% vs. 29%, P less than 0.01), which also is a 5-deiodination step. In conclusion, our data show that CRF profoundly changes the kinetics of all iodothyronines studied. Furthermore, our data are compatible with the existence of more than one 5'-deiodinase as well as more than one 5-deiodinase in man.     

Renal handling of thyroxine, 3,5,3'- and 3,3',5'-triiodothyronine, 3,3'- and 3',5'-diiodothyronine in man

The 24-h urinary excretion and renal clearance of thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), and 3',5'-diiodothyronine (3',5'-T2) were measured in 17 healthy subjects. The median urinary excretion was (pmol/24h) T4: 1242, T3: 828, rT3: 12.9, 3,3'-T2: 331, and 3',5'-T2: 5.8. The corresponding renal clearances were in median (ml/min) T4: 31, T3: 133, rT3: 15, 3,3'-T2: 683, and 3',5'-T2: 4.5. The clearances differed mutually (P less than 0.01) as well as from the creatinine clearance (P less than 0.01) which was in median 87 ml/min. Thus, all iodothyronines studied were subject to tubular transport mechanisms besides glomerular filtration. The 3 iodothyronines with 2 iodine atoms in the phenolic ring of the thyronine molecule, T4, rT3 and 3',5'-T2, were mainly tubularly reabsorbed, whereas those with only one iodine atom in the phenolic ring, T3 and 3,3'-T2, were mainly tubularly secreted. It might be hypothesized that the number of iodine atoms in the phenolic ring determines the direction of the tubular transport (presence of 2 iodine atoms is associated with tubular reabsorption, and of one iodine atom with secretion), whereas the rate of tubular transport decreases with decreasing number of iodine atoms in the tyrosylic ring.     

Concentrations of seven iodothyronine metabolites in brain regions and the liver of the adult rat

The concentrations of the iodothyronine metabolites T(4), T(3), 3,5-diiodothyronine (3,5-T(2)), 3,3'-diiodothyronine (3,3'-T(2)), reverse T(3) (rT(3)), 3,3'-T(2) sulfate (3,3'T(2)S), and T(3) sulfate (T(3)S) were measured in 12 regions of the brain, the pituitary gland, and liver in adult male rats. Quantification of iodothyronine was performed by RIA following a newly developed method of purification and separation by HPLC. 3,5-T(2), 3,3'-T(2), rT(3) and T(2)S were detectable in the low femtomolar range (20-200 fmol/g) in most areas of the rat brain. T(3)S was detectable only in the hypothalamus. The concentrations of T(3) and T(4) were approximately 20- to 60-fold higher, ranging between 1 and 6 pmol/g. There was a significant negative correlation between the activities of inner-ring deiodinase and T(3) concentrations across brain areas. In the liver, 3,5-T(2), rT(3), and T(3)S were measurable in the low femtomolar range, whereas 3,3'-T(2) and 3,3'T(2)S were not detectable. 3,5-T(2) and 3,3'-T(2) were not detectable in mitochondrial fractions of the brain regions. Tissue concentrations of 3,5-T(2) exhibited a circadian variation closely parallel to those of T(3) in the brain regions and liver. T(3) was not a substrate for outer-ring deiodination under different experimental conditions; thus, it remains unclear which substrate(s) and enzyme(s) are involved in the production of 3,5-T(2). These results indicate that five iodothyronine metabolites other than T(3) and T(4) are detectable in the low femtomolar range in the rat brain and/or liver. The physiological implications of this finding are discussed.     

The effects of Soybean Diet on Thyroid Hormone and Thyrotropin Levels in Aging Rats

OBJECTIVE: To estimate the effect of soybean diet on serum level of thyroid hormone, its metabolites and thyrotropin (TSH) during aging in rats. METHODS: Male Donryu rats were fed laboratory chow containing 40 (Group A) or 10 volume percent (Group B) soybean protein, while controls (Group C) received regular laboratory chow. Groups of 10 animals of each groups were sacrificed by decapitation at the age of 12, 18, 24 and 30 months. Serum total thyroxine (T4), free thyroxine (FT4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine (3,3'-T2) and TSH concentrations were measured by specific radioimmunoassays. RESULTS: In Group A the level of T3 decreased significantly at from the age of 18 months, while in other groups such decrease was found only from the age of 24 months. Such changes were closely resembled by these in the level of 3,3'-T2, while inverse changes were observed in the level of rT3 which was increased in Group A from the age of 18 months and in the other groups from the age of 24 months. Serum T4 and FT4 level was decreased in all groups at the age of 30 months and no changes were observed in the level of TSH. CONCLUSIONS: The findings suggest that the level of T4, FT4 and T3 with its metabolite 3,3'-T2 stepwise decreased with aging, while that of rT3 showed inversely and increase. These changes were influenced by the content of soybean protein in the diet, the most rapid changes being found in the group with the high content of such protein.     

Thyromimetic effects of 3,5-dimethyl,3'-isopropyl thyronine (DIMIT) and 3,5-diethyl,3'-isopropyl thyronine (DIET) in various tissues of the rat

We have examined the effects of treatment of the rat with 3,5-dimethyl-3'-isopropyl thyronine (DIMIT) and 3,5-diethyl-3'-isopropyl thyronine (DIET) on serum thyrotropin (TSH) concentration, heart weight, hepatic outer ring (5'-) monodeiodination of T4 to T3, cardiac outer ring monodeiodination of 3'-5'-diiodothyronine (3',5'-T2) to 3'-monoiodothyronine (3'-T1), and cerebral cortical monodeiodination of 3,5-T2 in the inner ring to 3-T1. Groups of four to seven rats were injected intraperitoneally either once a day or at eight-hour intervals for three days with saline or thyronines. Serum TSH was measured by radioimmunoassay. The various monodeiodinations were studied in homogenates of the tissues at optimal pH (7.35 for outer ring monodeiodinations and 8.0 for inner ring monodeiodination) and temperature (37 degrees C) in the presence of an excess of dithiothreitol. DIMIT was clearly active in all thyromimetic effects studied. It was about 10% as active as T4 in suppression of TSH. It stimulated hepatic monodeiodination of T4 to T3 in a similar manner to T4. DIMIT was also more comparable to T4 than to T3 in effect on heart weight and cardiac metabolism of 3',5'-T2 to 3'-T1. It was highly active, apparently more so than either T4 or T3, in cerebral cortical stimulation of metabolism of 3,5-T2 to 3-T1. In distinction to DIMIT, DIET had very little thyromimetic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Handling of iodothyronines by the liver and kidney in patients with chronic liver disease

Possible arterio-venous gradients of T4, T3, rT3 and 3,3'-diiodothyronine (3,3'-T2) across the liver and the kidneys were measured in 9 patients with varying degrees of liver failure undergoing diagnostic catheterization. Plasma iodothyronine levels were measured in peripheral, hepatic and renal veins before and at 10-min intervals until 60 min after iv injection of 400 micrograms of TRH. In 2 patients estimated hepatic plasma flow and effective renal plasma flow were determined as well. In these 2 patients, no significant differences between iodothyronine levels in arterial and peripheral venous plasma were found. T4 and T3 levels were not significantly different between peripheral, renal and hepatic veins. Hepatic vein rT3 and 3,3'-T2 concentrations were 10.7 +/- 8.3% (mean +/- SD, P less than 0.005) and 36 +/- 18% (P less than 0.001) lower than those in the peripheral vein (N = 9). Renal vein rT3 was just (6.2 +/- 7.5%, P less than 0.05) lower than rT3 in peripheral vein, whereas 3,3'-T2 was not different between the two veins. Estimates of hepatic and renal plasma flow were in agreement with values from the literature. On the basis of these data approximate hepatic clearance rates of 110 and 380 1/day for rT3 and 3,3'-T2 and a renal clearance rate of about 35 1/day for rT3 were calculated. Sixty min after TRH, plasma T3 was increased to 147 +/- 56% (P less than 0.05) and 3,3'-T2 in peripheral plasma was increased to 142 +/- 36% (P less than 0.025), whereas plasma T4 and rT3 did not change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of three-day oral cholecystography on serum iodothyronines and TSH concentrations: comparison of the effects among some cholecystographic agents and the effects of iopanoic acid on the pituitary-thyroid axis

The effects of repeated doses of oral cholecystographic agents on serum thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and thyrotrophin (TSH) concentrations were studied in 37 euthyroid male subjects. Iobenzamic acid, tyropanoic acid, iopanoic acid, and ipodate sodium, in a dosage of 3 g for 3 days, respectively, induced a significant decrease in serum T3 and an increase in rT3 within 24 h after the initial dose, followed by an increase in TSH and a slight increase in T4. The extent of the changes in rT3 varied between the agents, ipodate causing the greatest change, but without any relation to the changes in T3 or T4. Responses of serum T4, T3, rT3 and TSH concentrations to exogenous thyrotrophin-releasing hormone (TRH) and bovine TSH were also studied before and after 3-day doses of iopanoic acid. In 11 subjects given iopanoic acid, the response to TSH to TRH (500 micrograms, iv) was increased but the T3 response was unchanged. A dose of TSH (10 U.S.P. units, im) caused a significant increase in serum T3 and a decrease in TSH concentrations in 5 subjects both before and after cholecystography. It is thus suggested that in euthyroid subjects given multiple doses of oral cholecystographic agents, (1) the primary and consistent events are the reciprocal changes of serum T3 and RT3, although the extent of the changes is not coordinately reciprocal; (2) the responsiveness of the pituitary thyrotrophs and thyroid to TRH is preserved; and (3) the high basal and TRH-induced TSH in the serum may be ascribed to the decrease in the serum T3 concentration.     

Thyronamines are isozyme-specific substrates of deiodinases

3-Iodothyronamine (3-T 1 AM) and thyronamine (T AM) are novel endogenous signaling molecules that exhibit great structural similarity to thyroid hormones but apparently antagonize classical thyroid hormone (T(3)) actions. Their proposed biosynthesis from thyroid hormones would require decarboxylation and more or less extensive deiodination. Deiodinases (Dio1, Dio2, and Dio3) catalyze the removal of iodine from their substrates. Because a role of deiodinases in thyronamine biosynthesis requires their ability to accept thyronamines as substrates, we investigated whether thyronamines are converted by deiodinases. Thyronamines were incubated with isozyme-specific deiodinase preparations. Deiodination products were analyzed using a newly established method applying liquid chromatography and tandem mass spectrometry (LC-MS/MS). Phenolic ring deiodinations of 3,3',5'-triiodothyronamine (rT3AM), 3',5'-diiodothyronamine (3',5'-T2AM), and 3,3'-diiodothyronamine (3,3'-T2AM) as well as tyrosyl ring deiodinations of 3,5,3'-triiodothyronamine (T3AM) and 3,5-diiodothyronamine (3,5-T2AM) were observed with Dio1. These reactions were completely inhibited by the Dio1-specific inhibitor 6n-propyl-2-thiouracil (PTU). Dio2 containing preparations also deiodinated rT(3)AM and 3',5'-T2AM at the phenolic rings but in a PTU-insensitive fashion. All thyronamines with tyrosyl ring iodine atoms were 5(3)-deiodinated by Dio3-containing preparations. In functional competition assays, the newly identified thyronamine substrates inhibited an established iodothyronine deiodination reaction. By contrast, thyronamines that had been excluded as deiodinase substrates in LC-MS/MS experiments failed to show any effect in the competition assays, thus verifying the former results. These data support a role for deiodinases in thyronamine biosynthesis and contribute to confining the biosynthetic pathways for 3-T 1 AM and T 0 AM.     

Phenolic and tyrosyl ring iodothyronine deiodination by the Caco-2 human colon carcinoma cell line

Thyroid hormone metabolism was studied in the human Caco-2 colon carcinoma cell line, which at confluence exhibits several functions of differentiated enterocytes. Cells were harvested two to 17 days after reaching confluence. Intact cells and homogenates were tested for deiodination of [125I]-labeled substrates. Small amounts of thyroxine (T4) were converted by homogenates to 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), and 1-, with no detectable production of 3,5,3'-triiodothyronine (T3) by homogenates or cells. rT3 was converted to 3,3'-T2 and 1- with an apparent Michaelis constant (Km) for rT3 of 24 nmol/L; 6-n-propyl-2-thiouracil (PTU) had a 50% inhibitory concentration of 30 nmol/L and abolished rT3 5'-deiodination at 1 mmol/L in the presence of 20 mmol/L dithiothreitol (DTT). T3 was deiodinated to 3,3'-T2 and 3'-monoiodothyronine (3'-T1) with an apparent Michaelis constant (Km) for T3 of 5.7 nmol/L; this reaction was not inhibited by 1 mmol/L PTU. Phenolic and tyrosyl ring deiodinating activities were maximal four and six days, respectively, after the cells reached confluence. Homogenates of cells grown in standard medium containing fetal calf serum had fivefold higher rT3 5'-deiodinating activity than cells grown in a serum-free defined culture medium, reflecting a fivefold difference in the apparent Vmax with no difference in the apparent Km for rT3. There was no difference in T3 5-deiodination rates in homogenates of Caco-2 cells grown in the two media until 12 days postconfluence, when cells grown in standard medium had higher activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Splanchnic extraction of 3,3'-diiodothyronine and 3',5'-diiodothyronine in hyperthyroidism

The splanchnic extraction of 3,3'-diiodothyronine (3,3'-T2) and 3',5'-diiodothyronine (3',5'-T2) was studied in 7 hyperthyroid patients and 20 normal subjects employing the hepatic venous catheterization technique. A significant net uptake by splanchnic tissues was found for both diiodothyronines . The fractional splanchnic extraction calculated as the arterio-hepatic venous plasma concentration difference divided by the arterial concentration was unaffected by hyperthyroidism as compared to normal values. There was a close positive correlation between the arterio-hepatic venous concentration difference and arterial concentration, 3,3'-T2: r = 0.988, and 3',5'-T2: r = 0.932 (P less than 0.001). The splanchnic extraction was nonsaturable at endogenous plasma concentrations of 3,3'-T2 up to at least 17.0 ng/dl and of 3',5'-T2 up to at least 15.2 ng/dl. The data suggest that the splanchnic extraction of 3,3'-T2 and 3',5'-T2 obeys first order kinetics, the fractional extraction being unaffected by hyperthyroidism. Furthermore, changes in the net splanchnic extraction of 3,3'-T2 and 3',5'-T2 do not seem to contribute to changes in circulating levels of these iodothyronines. It is suggested that tissues other than the liver contribute significantly to the deiodination process both in normal and in hyperthyroid man.     

Variation in the release of thyroxine, triiodothyronine and growth hormone in response to thyrotrophin releasing hormone during development of the domestic fowl

In newly hatched chicks, TRH administration was followed by increased circulating concentrations of growth hormone (GH), thyroxine (T4) and 3,3',5-triiodothyronine (T3). Little change in the circulating concentration of 3,3',5'-triiodothyronine (rT3) was however observed. The effect of TRH on the circulating concentration of GH was not found in chick embryos at 17 or 19 days of incubation but was observed during pipping. The T4 response was maximal in newly hatched chicks and present in 17 and 19 days embryos. The magnitude of the T3 responses increased with age while that of T4 decreased.     

SERUM LEVELS OF T4, T3, REVERSE T3, 3,3'-DIIODOTHYRONINE AND 3',5'-DIIODOTHYRONINE IN OBESITY, BEFORE AND AFTER JEJUNO-ILEAL BYPASS

Serum T4, 3,5,3′-triiodothyronine (T3), 3,3′,5′-triiodothyronine (reverse T3, rT3), 3,3′-diiodothyronine (3,3′-T2), 3′,5′-diiodothyronine (3′,5′-T2) and thyrotrophin (TSH) levels were studied in nineteen obese patients before and 6, 12, and 18, months after a jejuno-ileal bypass. Before surgery, the obese patients had increased serum T3 levels compared with a group of lean, matched controls (median: 1·94 nmol/1 v. 1·44 nmol/l, P < 0·01). Serum T3 decreased to normal (1·64 nmol/l) 18 months after surgery. A slight decrease was also observed in serum 3,3′-T2 levels, whereas progressive reductions in serum concentrations of rT3 and 3′,5′T2 occurred. Eighteen months postoperatively the serum levels of rT3 and 3′,5′-T2 had decreased from 0·676 nmol/l to 0·430 nmol/l (P < 0·02) and 55–2 pmol/l to 40.0 pmol/l (P < 0·01), respectively, and the values at 18 months were also reduced compared with the control group [0·722 nmol rT3/1 (P < 0·01), 51·4 pmol 3′,5′-T2/1 (P < 0·01)]. Concomitant with the decrease in serum levels of the iodothyronines, serum TSH concentrations increased from 0 μu/ml to 0·9 μu/ml (P < 0·01).     

3'-5'-Diiodothyronine in health and disease: studies by a radioimmunoassay.

A simple, reproducible, and highly specific RIA has been developed for measurement of 3',5'-diiodothyronine ((3',5'-T2) in unextracted serum. Interference in binding of radioactive 3',5'-T2 to anti-3',5'-T2 by serum proteins was minimized by using 0.4 M phosphate buffer (pH 6.2) and merthiolate. The detection threshold of the RIA was 2.5 ng/100 ml. Recovery of nonradioactive 3',5'-T2 added to serum averaged 99%. T4, T3, and rT3 cross-reacted with 3',5'-T2-binding sites on anti-3',5'-T2 antibody only to the extent of 0.0025, less than 0.0004, and 0.22%, respectively. 3'-Monoiodothyronine cross-reacted 1.7%. Serum 3',5'-T2 concentrations were (mean +/- SD) 6.4 +/- 2.4 ng/100 ml in 53 normal subjects, 4.2 +/- 3.5 ng/100 ml in 7 hypothyroid patients, 14.9 +/- 7.7 ng/ml in 25 patients with hepatic cirrhosis, and 14.3 +/- 5.3 ng/100 ml in 31 newborns' cord blood sera. The values in each of the latter four groups were significantly different from normal. The mean serum 3',5'-T2 concentration of 7.7 +/- 2.5 ng/ml in eight subjects in the third trimester of pregnancy did not differ significantly from normal at a time when serum T4 and T3 were clearly elevated. Oral administration of 300 microgram rT3 to 9 normal subjects led to a mean maximal increase in serum 3',5'-T2 concentration of 45% at 1 h. Total fasting in 3 obese subjects was associated with a significant increase in serum 3',5'-T2 from 8.6 to 16.3 ng/100 ml at 6-8 days; serum rT3 increased similarly, while serum T3 decreased and T4 did not change. Administration of dexamethasone (2 mg also associated with nearly parallel increases in serum 3',5'-T2 and rT3 and a decrease in serum T3. 3',5'-T2 concentrations were also measured in amniotic fluids at different stages of gestation; the mean value of 15.2 ng/100 ml at 15-20 weeks gestation was significantly higher than that of 5.8 ng/ml at 33-40 weeks gestation. Pronase hydrolysates of 9 autopsy specimens of normal thyroid glands contained (mean +/- SD) 350 +/- 144 microgram T4 and 0.24 +/- 0.15 microgram 3',5'-T2/g wet wt. On the basis of these data and those available for MCRs of 3',5'-T2 and T4, it was estimated that thyroidal secretion contributes less than 1% of 3',5'-T2 measured in serum of normal man. The various data suggest that: 1) 3',5'-T2 is a normal component of human serum; 2) almost all 3',5'-T2 in human serum derives from extrathyroidal sources; and 3) changes in serum 3',5'-2 generally parallel those in rT3.     

Monodeiodination of triiodothyronine and reverse triiodothyronine during low and high calorie diets

The impact of varying caloric intake on peripheral monodeiodination and plasma disposal of T3, rT3, and the three diiodothyronines (T2) was studied in five normal subjects while they were consuming a low calorie diet (1200 Cal/day) and again while receiving a high calorie diet (3600 Cal/day). Toward the end of each diet period 240 nmol 3,3'-T2 (126 micrograms) and 80 nmol 3',5'-T2 (42 micrograms) were infused for 7 h, and a bolus injection of 137 nmol 3,5-T2 (72 micrograms) was followed by a 12-h infusion of 69 nmol 3,5-T2 (36 micrograms) and 111 nmol rT3 (72 micrograms) on another day. [125I]T3 (30 muCi) was injected on the third day. The T2 and rT3 concentrations were measured by RIA during the 2 days of infusion, and the serum disappearance of [125I]T3 was studied by immunoprecipitation and trichloroacetic acid precipitation of the labeled T3. Four to 5% of the plasma disposal of T3 was accounted for by 3'-monodeiodination, and 36-39% by 5-monodeiodination. Increasing caloric intake resulted in a higher overall plasma disposal rate of T3, but no change in the percentage of T3 metabolized by monodeiodination pathways. In contrast, 5'-monodeiodination accounted for 21% of the total plasma disposal of rT3 during the low calorie diet and 45% during the high calorie intake. This increase in 5'-monodeiodination of rT3 was at the expense of alternative pathways of disposal. A marked increase in the plasma clearance rate of 3,5-T2 was also found during the high calorie diet, indicating that the level of caloric intake affects pathways of metabolism other than outer ring monodeiodination. These studies emphasize the important role played by diet in the regulation of peripheral thyroid hormone metabolism through modulating outer ring monodeiodination, and that overnutrition changes other pathways of iodothyronine metabolism as well.     

Fecal and urinary excretion of six iodothyronines in the rat

Fecal and urinary excretion rates of six iodothyronines were assessed in the rat maintained under normal steady state physiological conditions, to gain a more comprehensive understanding of the mechanisms of control of normal thyroid hormone economy and metabolism. Groups of young adult male rats were injected with trace doses of T4, T3, rT3, 3,3'-diiodothyronine (T2), 3',5'-T2, or 3'-monoiodothyronine, each labeled with 125I, and feces and urine were collected separately for up to 10 days. Pooled fecal pellets were homogenized in saline, extracted in ethanol, evaporated under vacuum, and reconstituted in NaOH. Fecal extracts and urine were chromatographed on Sephadex G25 columns under conditions providing quantitative separations of components of interest. A new technique was also developed, based on a model of the in vitro extraction and measurement process, to correct chromatographic results for possible variable recoveries and possible artifactious degradation of radioactively labeled components. No iodothyronines or their conjugates were excreted in urine; all radioactivity was in the form of iodide. In feces, about 30% of the [125I]T3 injected was excreted as T3; and 24% of the [125I]T4 injected was excreted as T4, plus 4% as T3. Together, these results imply that about 24% of endogenous T4 production is excreted as T4 and 76% is irreversibly metabolized; and for T3, about 30% of endogenous T3 production is excreted as T3 and 70% is degraded. For the nonhormonal iodothyronines, about 6% of injected monoiodothyronine, 3% of injected 3',5'-T2, 2% of injected 3,3'-T2, and less than 1% of injected rT3 were excreted in feces as such, indicating that these substances are nearly completely deiodinated in vivo. Very little (1-7%) iodide was excreted as such in feces, which also were devoid of measurable conjugates. An open question is whether the substantial wastage of thyroid hormones in feces represents poor hormone economy in the usually accepted sense or a functional property of overall thyroid hormone regulation.     

Acute effects of thyroid hormone analogs on sodium currents in neonatal rat myocytes.

We previously reported that T3(3,3',5-triiodo-L-thyronine) acutely increases sodium currents (INa) in neonatal rat myocytes. Here we compare the effects of several thyroid hormone analogs, including T4(3,3',5,5'-tetraiodo-L-thyronine), rT3(3,3',5'-triiodo-L-thyronine), D-T3(3,3',5-triiodo-D-thyronine), 3,5-T2(3,5-diiodo-L-thyronine), DIT (3,5-diiodo-L-tyrosine), MIT (3-monoiodo-L-tyrosine), tetrac (3,3',5,5'-tetraiodo-thyroacetic acid), triac (3, 3',5-triiodo-thyroacetic acid), and tyrosine, on INa in cultured neonatal rat myocytes (n ranged from 9 to 28 for each comparison). T4, T3, 3,5-T2, and DIT (10 n m) all increased current density relative to control to a similar degree: to 1.22+/-0.2, 1.21+/-0.03, 1.16+/-0.02 and 1.16+/-0.03, respectively, P<0.05. In contrast, thyroid hormone analogs with an altered side group of the inner iodophenyl ring, including tetrac, triac, and D-T3, had no effect on INa nor did rT3, MIT or tyrosine. Pretreatment with rT3 inhibited the effects of T4, T3, 3,5-T2, and DIT. Conversely, the dose-dependent inhibitory effect of amiodarone, an iodinated benzofuran derivative that antagonizes thyroid hormone actions, on INa was blocked when myocytes were pretreated with T3(100 n m, n=3), suggesting an interaction of T3 with amiodarone. The enhancement of INa by T3 and 3, 5-T2 could not be blocked by propranolol, suggesting that the effects are not mediated through beta -adrenergic signaling pathways. In conclusion, the present results suggest that the acute effects of thyroid hormone and analogs on cardiac INa are mediated by a non-genomic thyroid hormone receptor with a unique structure-activity relationship.     

Characterization of human liver thermostable phenol sulfotransferase (SULT1A1) allozymes with 3,3',5-triiodothyronine as the substrate.

Sulfotransferase 1A1 (SULT1A1) (thermostable phenol sulfotransferase, TS PST1, P-PST) is important in the metabolism of thyroid hormones. SULT1A1 isolated from human platelets displays wide individual variations not only in the levels of activity, but also in thermal stability. The activity of the allelic variant or allozyme SULT1A1*1, which possesses an arginine at amino acid position 213 (Arg213) has been shown to be more thermostable than the activity of the SULT1A1*2 allozyme which possesses a histidine at this position (His213) when using p-nitrophenol as the substrate. We isolated a SULT1A1*1 cDNA from a human liver cDNA library and expressed both SULT1A1*1 and SULT1A1*2 in eukaryotic cells. The allozymes were assayed using iodothyronines as the substrates and their biochemical properties were compared. SULT1A1*1 activity was more thermostable and more sensitive to NaCl than was SULT1A1*2 activity when assayed with 3,5,3'-triiodothyronine (T(3)). Sensitivities to 2,6-dichloro-4-nitrophenol (DCNP) and apparent K(m) values for SULT1A1*1 and for SULT1A1*2 with iodothyronines were similar. Based on K(m) values, the preferences of these SULT1A1 allozymes for iodothyronine substrates were the same (3,3'-diiodothyronine (3,3'-T(2))>3', 5',3-triiodothyronine (rT(3))>T(3)>thyroxine (T(4))>3,5-diiodothyronine (3,5-T(2))). SULT1A1*1 activity was significantly higher than the SULT1A1*2 activity with T(3) as the substrate. Potential differences in thyroid hormone sulfation between individuals with predominant SULT1A1*1 versus SULT1A1*2 allozymes are most likely due to differences in catalytic activity rather than substrate specificity.     

Deiodination of iodothyronine sulfamates by type I iodothyronine deiodinase of rat liver

The substrate behavior of synthetic N-sulfonated iodothyronines (iodothyronine sulfamates, TiNS) for the type I deiodinase was compared with that of the naturally occurring 4'-O-sulfonated iodothyronines (iodothyronine sulfates, TiS), which have been shown to be deiodinated 40-200 times more efficiently than the native iodothyronines. Deiodination was studied in incubations of rat liver microsomes with unlabeled or 3' (5')-125I-labeled T4NS, rT3NS, T3NS, and 3,3'-T2NS at 37 C and pH 7.2 in the presence of 5 mM dithiothreitol. Reaction products were analyzed by RIA or Sephadex LH-20 and HPLC. Kinetic studies were performed under initial reaction rate conditions to determine the apparent Michaelis Menten (Km) constants and maximum velocity values. In contrast to T4S, which is converted only by inner ring deiodination (IRD), T4NS underwent both IRD and outer ring deiodination (ORD), similar to T4, but more rapidly. At 10 nM T4NS substrate, T3NS was the major product observed, while no rT3NS accumulated due to its rapid conversion to 3,3'-T2NS. At least one third of the 3,3'-T2NS was converted by IRD, unlike 3,3'-T2 which is a pure ORD substrate. The type I deiodination efficiencies of T4NS IRD and ORD were 17-fold higher than with T4, mainly due to approximately 32-fold lower apparent Km values. Deiodination of rT3, the preferred type I substrate, was not improved by sulfamation. T3NS and 3,3'-T2NS were deiodinated 4-10 times more efficiently than T3 and 3,3'-T2, respectively, due to 2- to 4-fold decreases in apparent Km values with a concomitant doubling of maximum velocity values. N-Sulfonation stimulates type I deiodination to a similar extent as other side-chain modifications that eliminate the positive charge of the nitrogen (e.g. iodothyroacetic acids). However, the effects are less dramatic than those induced by 4'-sulfation with respect to both efficiency and specificity of the catalytic process.     

Metabolism of 3,3'-diiodothyronine in rat hepatocytes: interaction of sulfation with deiodination

Production of 3,3'-diiodothyronine (3,3'-T2) is an important step in the peripheral metabolism of thyroid hormone in man. The rapid clearance of 3,3'-T2 is accomplished to a large extent in the liver. We have studied in detail the mechanisms of this process using monolayers of freshly isolated rat hepatocytes. After incubation with 3,[3'-125I]T2, chromatographic analysis of the medium revealed two major metabolic routes: outer ring deiodination and sulfation. We recently demonstrated that sulfate conjugation precedes and in effect accelerates deiodination of 3,3'-T2. In media containing different serum concentrations the cellular clearance rate was determined by the nonprotein-bound fraction of 3,3'-T2. At substrate concentrations below 10(-8) M 125I- was the main product observed. At higher concentrations deiodination became saturated, and 3,3'-T2 sulfate (T2S) accumulated in the medium. Saturation of 3,3'-T2 clearance was found to occur only at very high (greater than 10(-6)M) substrate concentrations. The sulfating capacity of the cells exceeded that of deiodination by at least 20-fold. Deiodination was completely inhibited by 10(-4) M propylthiouracil or thiouracil, resulting in the accumulation of T2S while clearance of 3,3'-T2 was little affected. No effect was seen with methimazole. Hepatocytes from 72-h fasted rats showed a significant reduction of deiodination but unimpaired sulfation. Other iodothyronines interfered with 3,3'-T2 metabolism. Deiodination was strongly inhibited by 2 microM T4 and rT3 (80%) but little by T3 (15%), whereas the clearance of 3,3'-T2 was reduced by 27% (T4 and rT3) and 12% (T3). It is concluded that the rapid hepatic clearance of 3,3'-T2 is determined by the sulfate-transferring capacity of the liver cells. Subsequent outer ring deiodination of the intermediate T2S is inhibited by propylthiouracil and by fasting, essentially without an effect on overall 3,3'-T2 clearance.