Triiodothyronine: a substrate for the thermostable and thermolabile forms of human phenol sulfotransferase

The enzyme(s) responsible for the sulfate conjugation of L-T3 in man has not been characterized. T3 sulfotransferase (T3-ST) activity was characterized in normal human liver tissue obtained during clinically indicated surgical resection. Subcellular distribution studies showed that the T3-ST activity was localized to the cytoplasmic fraction. This finding raised the possibility that T3-ST activity might be similar to the 2 previously identified forms of cytoplasmic phenol sulfotransferase (PST) found in human tissue. A thermostable (TS) form of PST catalyzes the sulfate conjugation of micromolar concentrations of p-nitrophenol, and a thermolabile (TL) form catalyzes the sulfate conjugation of dopamine and other monoamines. Thermal stability and enzyme inhibitor experiments showed that T3-ST activity in pooled liver homogenates was very similar to the TS form of PST. The apparent similarity of T3-ST to TS PST was studied further by measuring T3-ST, TS PST, and TL PST activities in 20 individual liver samples. T3-ST activities correlated significantly with TS PST activities (r = 0.939; P less than 0.001) measured with p-nitrophenol, but not with TL PST activities (r = -0.118; P greater than 0.6) measured with dopamine. However, sulfation of T3 by the TL form of the enzyme might have been masked by the 18-fold higher specific activity of TS than TL PST in human liver homogenates. When the two forms of PST were separated by ion exchange chromatography, T3 was found to be a substrate for both the TS and TL forms of PST. "True" Km values for T3 were similar for TS and TL PST (81 and 127 microM, respectively).     

Transport of thyroxine and 3,3',5-triiodothyronine in human umbilical vein endothelial cells

The prerequisite for the uptake of thyroid hormone (TH) in peripheral tissues is the exit of TH from the bloodstream. The first step in this process is transport across the endothelium. Little is known about this important step in TH physiology. Therefore, we aimed to characterize the TH transport processes across the endothelium using human umbilical vein endothelial cells as a model. Transport studies showed rapid uptake of 1 nm [(125)I]T(3) and [(125)I]T(4) in these cells. The apparent Michaelis constant value for [(125)I]T(3) uptake was about 1 microm, and the IC(50) for T(4) inhibition of T(3) uptake was about 3 microm. The aromatic amino acids phenylalanine, tyrosine, and tryptophan and the L-type amino acid transporter-specific ligand 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid did not inhibit [(125)I]T(3) or [(125)I]T(4) uptake. Verapamil was capable of reversibly reducing transport of [(125)I]T(3) and [(125)I]T(4). Human umbilical vein endothelial cells incubated with the affinity label BrAcT(3) resulted in a labeling of multiple proteins, which are probably protein disulfide isomerase related. Extrapolating our findings to the endothelial lining of blood vessels suggests that T(3) and T(4) uptake is mediated by the same transport system. Because TH transport characteristics do not correspond to known TH transporters, further studies are required to identify the TH transporter protein(s) at the molecular level. Possible candidates may be widely expressed Na(+)-independent transporter proteins.     

Opposite effects of dexamethasone on serum concentrations of 3,3',5'-triiodothyronine (reverse T3) and 3,3'5-triiodothyronine (T3).

Dexamethasone, 2 mg every 6 hours for 4 doses, was given to 4 hypothyroid patients receiving treatment with synthetic thyroxine (T4) and to 8 untreated hyperthyroid patients with Graves' disease, and serum concentrations of thyroid hormones were measured by radioimmunoassays. Serum concentration of 3,3'5'-triiodothyronine (reverse T3, rT3) increased appreciably within 8 hours after the first dose of dexamethasone, was maximum at 24-32 hours after beginning dexamethasone, and remained elevated for about 24 hours after discontinuing the steroid. The mean baseline serum rT3 was 58 ng/per 100 ml in treated hypothyroid patients and 119 ng per 100 ml in patients with Graves' disease; the corresponding maximal post-dexamethasone serum rT3 values were 87 and 170 serum concentration of 3,3',5-triiodothyronine (T3) decreased. The decrease in serum T3 was significant at about 24 hours after beginning dexamethasone and was maximal at about 30 hours in both groups of cases under study. The decrease in serum T3 persisted in treated hypothyroid cases for about 24-48 hours and in Graves' disease cases as long as studied, at least 5 days after discontinuing hexamethasone. The changes in serum rT3 and T3 could not be attributed to the effect of dexamethasone on serum protein binding of the iodothyronines because the dialyzable fractions of rT3 and T3 following steroid administration were not different from those before it. Serum T4 did not change appreciably in treated hypothyroid cases, but decreased in Graves' disease cases from a mean baseline value of 23.5 mug per 100 ml to 18.4 mug per 100 ml 3 days after beginning dexamethasone. In addition, 3 hyperthyroid cases were studied before, during, and after administration of dexamethasone, 2 mg every 6 h for 5 days. Serum rT3 increased again as noted above and the increase persisted until about 24 hours after the last dose of the steroid. Serum T3 decreased considerably and remained decreased as long as studied, at least 4 days after discontinuing the steroid. Serum T4 decreased appreciably in 2 of the 3 cases studied. The data suggest that 1) conversion of T4 to T3 and to rT3 may occur via two distinct pathways in the metabolism of T4; 2) the changes in serum rT3 and T3 observed in our study may be due in part at least to a steroid-induced 'shift' in the metabolism of T4 whereby conversion of T4 to T3 is diminished and that to rT3 is enhanced; 3) in addition to the effect on peripheral metabolism of T4, steroids appear to reduce the circulating thyroid hormones in Graves' disease by another mechanism, probably by reduction in thyroid secretion.     

Thyroxine and 3,3',5-triiodothyronine are glucuronidated in rat liver by different uridine diphosphate-glucuronyltransferases

Male Wistar rats were treated with 50 mg 3,3',4,4'-tetrachlorobiphenyl (TCB)/kg BW or vehicle. After 4 days, the livers were isolated and perfused for 90 min with 2 nM [125I]T3 or 10 nM [125I]T4 in Krebs-Ringer medium containing 1% albumin. Deiodination and conjugation products and remaining substrates were determined in bile and medium samples by Sephadex LH-20 chromatography and HPLC. TCB treatment did not affect hepatic uptake and metabolism of T3. However, biliary excretion of T4 glucuronide was strongly increased by TCB, resulting in an augmented T4 disappearance from the medium, although initial hepatic uptake of T4 was not altered. Measurement of the microsomal UDP-glucuronyltransferase (UDPGT) activities confirmed that T4 UDPGT was induced by TCB, whereas T3 glucuronidation was unaffected. T3 UDPGT activity showed a discontinuous variation, which completely matched the genetic heterogeneity in androsterone glucuronidation in Wistar rats. These results indicate that different isozymes catalyze the glucuronidation of T3 and T4.     

Identification of 3,3'-diiodothyroacetic acid sulfate: a major metabolite of 3,3',5-triiodothyronine in propylthiouracil-treated rats

The sulfate conjugate 3, [3'-125I] diiodothyroacetic acid (3,3'-TA2S) was discovered in plasma, and occasionally in bile, of 6-propyl-2-thiouracil-treated rats after administration of [125I]T3. The identification of this T3 metabolite was based on the following evidence: 1) the compound co-eluted in two different HPLC systems with synthetic 3,3'-TA2S; 2) its chromatographic behavior on Sephadex LH-20 was characteristic for a conjugated iodothyronine derivative; and 3) the metabolite was hydrolyzed by arylsulfatase and the liberated product comigrated with synthetic 3,3'-TA2 on HPLC. Marked accumulation of 3,3'-TA2S was observed only in rats with impaired type I deidodinase activity but not in controls. Furthermore, plasma and biliary 3,3'-TA2S levels varied with the experimental conditions such as anesthesia, i.e. both were increased in ketamine-anesthetized over pentobarbital-anesthetized animals. It was not possible to indicate the exact pathway through which 3,3'-TA2S is generated from T3; neither is it known how much of T3 is actually metabolized via 3,3'-TA2S. However, the significant plasma 3,3'-TA2S levels, even in unanesthetized animals, illustrate the physiological relevance of this T3 metabolite.     

The relative distribution of thyroxine, triiodothyronine and 3,3',5'-(reverse)-triiodothyronine in various fractions of thyroglobulin

Thyroglobulin fractions rich and poor in new thyroglobulin were separated by means of DEAE-cellulose chromatography of dog thyroid extracts and by zonal ultracentrifugation in a sucrose gradient of guinea pig thyroid extract incubated at low temperature. The distrubtion of thyroxine, triiodothyronine and 3,3',5'-(reverse)-triidothyronine in hydrolysates of the different fractions was estimated by radioimmunoassays. Following DEAE-cellulose chromatography there was a small but statistically significant increase in T4/T3 ratio in thyroglobulin fractions eluted at high ionic strength--that is fractions relatively rich in stable iodine but poor in fresh thyroglobulin. There was no differences in the T4/rT3 ratios between the different fractions. The ratios between iodothyronines were almost identical in the various thyroglobulin fractions following zonal ultracentrifugation in a sucrose gradient of cold treated guinea pig thyroid extract. These findings lend no support to the possibility that a relatively high content of triiodothyronines in freshly synthesized thyroglobulin modulates the thyroid secretion towards a preferential secretion of triiodothyronine and 3,3',5'-(reverse)-triidothyronine at the expense of the secretion of thyroxine.     

Metabolic clearance and production rates of 3,3',5-triiodothyronine in hyperthyroid, euthyroid, and hypothyroid subjects.

To further elucidate the peripheral metabolism of rT3 and to determine if rT3 production rates vary directly with thyroid function, we measured the disappearance of [125I]rT3 in thyrotoxic and hypothyroid subjects as well as in athyreotic patients maintained eumetabolic on exogenous T4. Kinetic parameters were determined by noncompartmental analysis, and serum concentrations of rT3 and T4 were determined by specific RIAs. In six hyperthyroid, seven euthyroid, and six hypothyroid subjects, the MCRs were 190.7 +/- 15.7, 111.7 +/- 13.2, and 71.8 +/- 7.0 liters/day kg, respectively (mean +/- SE). Production rates (PR) for these same groups were 271.3 +/- 40.5, 51.7 +/- 9.1, and 4.3 +/- 0.6 micrograms/day/70 kg. The observed differences in MCR and PR among the three study groups were highly significant (P less than 0.002). These data indicate that in comparison to euthyroid subjects, rT3 PR and MCR are increased in thyrotoxic and decreased in hypothyroid individuals.     

Inhibition of uptake of thyroid hormone into rat hepatocytes by preincubation with N-bromoacetyl-3,3',5-triiodothyronine

To investigate whether affinity coupling of N-bromoacetyl-T3 (BrAcT3) to the T3 membrane carrier results in an inhibition of transport of T3 into the cell, rat hepatocytes in monolayer were incubated for 2 h at 21 C with 1.3 mumol/liter BrAcT3 in medium without protein. After extensive washing, cells were incubated during 20 h at 37 C with [125I]T3 in medium with 0.5% BSA, and products in supernatants were analyzed by LH-20 column chromatography. In addition the apparent affinity constant (Km) and maximal uptake velocity (Vmax) of the high affinity uptake process were estimated using 1 min incubations of hepatocytes with various concentrations of T3. In control experiments (i.e. without BrAcT3 affinity coupling) about 57% of the added T3 was cleared from the medium and further metabolized, 85% of the cleared T3 reappeared in the medium as I-, 15% as conjugates. Addition of propylthiouracil during the 20 h incubation with T3 strongly inhibited deiodination, without a change in T3 clearance. Because T3 is sulfated before deiodination, a concomitant rise in conjugates was observed. Addition of ouabain to control cells during the 20 h incubation with T3 strongly inhibited uptake, with a parallel decrease in I- and conjugate formation. After affinity coupling of BrAcT3, T3 clearance was inhibited (by 30% P less than 0.001). Since I- production was more depressed (by 73%) than T3 clearance, with some rise in conjugate formation (P less than 0.001), inhibition of deiodinase by BrAcT3 also took place. The effects of BrAcT3 and ouabain on uptake of T3 appeared to be additive as were the effects of propylthiouracil and BrAcT3 on deiodination. After affinity coupling of BrAcT3, the Km of T3 uptake did not change significantly; however Vmax was 54% lower (P less than 0.025) indicating a noncompetitive inhibition of the transport system. Preincubation of the cells with N-acetyl-T3 does not alter the characteristics of uptake of T3 by rat hepatocytes as compared to controls, indicating that no binding of this compound occurs. It is concluded that preincubation of hepatocytes with BrAcT3 diminished I- formation from T3; 50% of this inhibition is due to decreased membrane transport and 50% by reduction of deiodination. Inhibition of membrane transport by BrAcT3 is substantiated by a 54% lower Vmax without a significant change in Km as compared to control. The effect of transport of thyroid hormone on metabolism stresses the importance of the membrane carrier in the translocation process.     

3,3',5'-Triiodothyronine (reverse T3) and 3,3',5-triiodothyronine (T3) in fetal and adult sheep: studies of metabolic clearance rates, production rates, serum binding, and thyroidal content relative to thyroxine.

To examine the mechanism(s) responsible for high serum concentration of 3,3',5'-triiodothyronine (reverse T3, rT3) and low serum concentration of 3,3',5-triiodothyronine (T3) in the fetus, we studied metabolic clearance rates (MCR) and production rates (PR) of rT3, T3, and thyroxine (T4) in adult nonpregnant sheep and sheep fetuses in utero. The mean fetal MCR-rT3 was significantly lower than that in adult sheep, and the mean fetal PR-rT3 significantly higher. The mean fetal MCR-T3 was higher than, and the mean fetal PR-T3 similar to that in adult sheep. The mean fetal MCR-T4 and PR-T4 were both significantly higher than the corresponding values in adult sheep. The ratios of mean PR-rT3 to PR-T4 (rT3/T4) were similar in fetal and adult sheep. However, the ratio of mean PR-T3 to PR-T4 (T3/T4) in the fetal sheep was much lower than that in the adult sheep. The low fetal MCR-rT3 was not attributable to high serum binding of rT3. On the basis of the thyroidal content and kinetics of iodothyronines, it was estimated that whereas thyroidal secretion may account for nearly all of serum T3 (or PR-T3) in the fetus and about 50% of serum T3 in adults, it accounts for only about 3% of the serum rT3 (or PR-rT3) in both fetal and adult sheep. The results suggest a) that elevated serum rT3 in the fetus is due to its decreased clearance and increased production by mono-deiodination of T4, and b) that low serum T3 in the fetus is due to its increased clearance and decreased production by mono-deiodination of T4. In addition, on the basis of discordant changes in the production of T3 and rT3 from T4, it appears that there may exist two separate, apparently specific, iodothyronine deiodinating activities--one cleaving the iodine atom at the 5'-position and the other acting in the iodine atom at the 5-position of the T4 molecule; 5'-iodothyronine deiodinating activity is apparently reduced in the fetus.     

Characterization of the 3,3',5-triiodo-L-thyronine-binding site on plasma membranes from human placenta

The binding of [125I]T3 to sites on human placenta plasma membranes was characterized, and the binding site was solubilized after affinity labeling with N-bromoacetyl-[125I]T3 (BrAc[125I]T3). Two classes of T3-binding sites were detected. One class has a high affinity (Kd = 2.0nM) and a low capacity (approximately 320 fmol/mg protein); the other has a low affinity (Kd = 18.5 microM) and a high capacity (approximately 2.2 pmol/mg protein). The binding sites were found to be specific for T3 in that other thyroid hormone analogs (D-T3, rT3, D-T4, and L-T4) were less effective or ineffective in displacing the bound [125I]T3. The affinity labeling ligand BrAc[125I]T3 was found to specifically label a protein with an apparent mol wt of 65,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The BrAc[125I]T3-labeled protein was solubilized with 2 mM 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate. The apparent mol wt of the labeled protein was between 140,000 and 150,000 by Sephadex-G-200 gel filtration. These data demonstrate that a high affinity binding site specific for T3 is present on plasma membranes from human placenta and that the binding site is a protein, most likely a dimer, with a native mol wt between 140,000 and 150,000.     

Effect of propranolol on extrathyroidal metabolism of thyroxine and 3,3',5-triiodothyronine evaluated by noncompartmental kinetics.

Kinetic studies of T4 and T3 using a noncompartmental approach were performed in seven patients with pretreatment severe hypothyroidism maintained on L-T4 replacement. Each subject received a combined tracer dose of labeled T4 and T3 as an iv bolus before and during peroral treatment with propranolol. Serum T4 was unchanged, while a significant decrease of 13% was found in serum T3. The disposal rates (DR) of T4 and T3 decreased significantly, and the ratio between the DR off T3 and the DR of T4, the conversion rate, was significantly reduced during propranolol treatment. The decrease in the DR of T4 suggests a reduction in the bioavailability of L-T4 during propranolol, possibly due to a decrease in intestinal absorption. The decrease in the conversion rate indicates a reduced extrathyroidal conversion of T4 to T3 during propranolol treatment.     

A new type of albumin with predominantly increased binding affinity for 3,3',5-triiodothyronine in a patient with Graves' disease

A new type of serum albumin, that shows a markedly enhanced binding activity for 3,3', 5-triiodothyronine (T3), a somewhat increased activity for thyroxine (T4), and a normal activity for 3,3', 5-triiodothyronine (rT3) is described. This albumin was found in a patient with Graves' disease. After successful subtotal thyroidectomy, the existence of abnormal binding activity for T3 was suspected in this patient because of persistently increased total T3 concentrations in spite of elevated thyrotropin levels. Although free T3 and T4 concentrations measured by radioimmunoassay using commercial tracer analogue kits were markedly increased, those measured by equilibrium dialysis were within normal ranges. Electrophoretic studies revealed that these abnormalities were due to the markedly increased T3 binding activity by the serum albumin; that for T4 was also slightly increased. Scatchard plot analysis revealed that the association constant (Ka) for T3 of the patient's albumin was 5.1 X 10(6)/M (normal pooled albumin; 6.2 X 10(5)/M), and those for T4 and rT3 were 5.2 X 10(6)/M and 2.7 X 10(6)/M, respectively (normal pooled albumin; 2.1 X 10(6)/M for both T4 and rT3). The increased binding of albumin to T3 and T4 was markedly inhibited by barbitone, and 8-anilino-1-naphthalene-sulfonic acid. These characteristic features, and erroneously high values of free T3 and T4 concentrations measured by tracer analogue kits were similar to those seen in patients with familial dysalbuminemic hyperthyroxinemia, which have been previously reported. These findings strongly suggest that this albumin is a new variant in various dysalbuminemic syndromes, and the abnormal binding of iodothyronines moieties in these syndromes are not biochemically identical.     

A rapid sporozoite ELISA using 3,3',5,5'-tetramethylbenzidine as the substrate chromogen

A modified version of the standard 2-site sporozoite enzyme-linked immunosorbent assay (ELISA) using 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate chromogen solution was adapted for rapid detection and identification of Plasmodium falciparum and P. vivax circumsporozoite (CS) proteins. The TMB-ELISA was evaluated using sporozoites from experimentally infected mosquitoes and laboratory colonized uninfected mosquitoes. Our data indicate comparable sensitivity levels between the TMB-ELISA and the standard ELISA, i.e., 50 P. falciparum or P. vivax sporozoites/50 microliters of test solution. Reactions inherent to the method were specific and background reactivity was minimal. The TMB-ELISA is rapid (1 hr), simple, uses a minimal amount of monoclonal antibodies, and is suitable for use in a wide range of laboratories.     

3,3',5'-Triiodothyronine inhibits collagen-induced human platelet aggregation.

To clarify further the activity of rT3, we examined the effect of rT3 on collagen-induced platelet activation as reflected by aggregation, serotonin release, and protein phosphorylation. rT3, T4, T3, and triiodothyroacetic acid inhibited collagen-induced platelet aggregation and serotonin release from platelets in a dose-dependent manner. However, thyronine did not inhibit collagen-induced platelet aggregation. The concentration at which rT3 inhibited by 50% collagen-induced platelet aggregation was 30 +/- 4 (mean +/- SE) mumol/L. rT3, T4, and T3 did not differ significantly in their abilities to inhibit platelet aggregation. Moreover, rT3 inhibited collagen-induced phosphorylation of the 20-kilodalton protein (myosin light chain) in platelets. In contrast, rT3 did not inhibit 12-O-tetradecanoylphorbol 13-acetate (TPA)- or thrombin-induced platelet aggregation and inhibited only minimally TPA-induced 40-kilodalton protein phosphorylation. These results suggest that rT3 inhibits collagen-induced platelet activation by inhibiting the activity of myosin light chain kinase and that it may be interesting to investigate some kinds of activity of rT3.     

Cytochrome P450 (CYP) 1A2, sulfotransferase (SULT) 1A1, and N-acetyltransferase (NAT) 2 polymorphisms and susceptibility to urothelial cancer

Purpose Arylamines are suspected to be the primary causative agent of urothelial cancer in tobacco smoke. In the human liver, arylamines are N-hydroxylated by a cytochrome P450 (CYP)1A2-catalyzed reaction, which produces a substrate for O-esterification that can be catalyzed by N-acetyltransferases (NAT) or sulfotransferases (SULT). Recently, several polymorphisms of CYP1A2, SULT1A1, and NAT2 that affect their activities have been reported.Methods In this study, 306 Japanese patients with urothelial transitional cell carcinoma and 306 healthy controls were compared for frequencies of CYP1A2, SULT1A1, and NAT2 genotypes.Results The frequencies of NAT2 intermediate or slow acetylator genotype were significantly higher in the urothelial cancer patients than in the healthy control subjects [odds ratio (OR)=1.49, 95% confidence interval (95% CI) 1.06–2.09, OR=3.23, 95% CI 1.72–6.08, respectively]. Stratifying by amount of smoking, among subjects who consumed >33.5 pack-years and carried the SULT1A1 *1/*1 or NAT2 slow acetylator genotype, the OR was 1.73 (95% CI 1.01–2.97) whereas it was 7.31 (95% CI 1.90–28.05) in non-smokers who carried the homozygous wild genotype, respectively. The relationships between CYP1A2, SULT1A1, and NAT2 polymorphisms and clinical findings including tumor differentiation, stage, and recurrence rate were analyzed. Only associations between NAT2 genotype and pathological findings were admitted, and the higher OR of NAT2 intermediate and slow acetylator genotype was more likely to present to a low-grade tumor (G1) among heavy-smokers.Conclusions Our results suggest that SULT1A1 *1/*1 and NAT2 slow acetylator genotypes might modulate the effect of carcinogenic arylamines contained in tobacco smoke, and that the modulation of NAT2 intermediate and slow acetylator genotype has a tendency to present a higher risk for highly differentiated tumors among heavy-smokers.     

URINARY EXCRETION OF 3,3',5'-TRIIODOTHYRONINE (REVERSE T3)

A simple and sensitive radioimmunoassay for reverse T3 in urine using small Sephadex G25 fine columns is described. The recovery of rT3 added to urine was on average 101.0 ± 4.2% (mean ± SEM). Detection limit was 4 pg/column. Urine excretion of rT3 (mean ± SD) was 72.0 ± 32.1 ng/24 hin 61 healthy euthyroid subjects with a slight increase with age (P < 0.05), 28.8 ± 18.2 ng/24 h in 12 hypothyroid patients and 183.6 ± 79.7 ng/24 h in 25 hyperthyroid patients.