A Nonradioactive DEHAL Assay for Testing Substrates, Inhibitors, and Monitoring Endogenous Activity.

Iodotyrosine deiodinase (DEHAL1) is a crucial enzyme in iodine homeostasis. Unbound mono- and diiodotyrosines are indispensable byproducts of thyroid hormone biosynthesis. Their iodine needs to be recovered to avoid iodine deficiency, as observed in genetic defects in DEHAL1. Despite its importance, the enzyme is rarely studied. The deiodination process can be monitored by radioactive tracers or via techniques involving mass spectrometry. However, isotope-labeled molecules are expensive, not always commercially available, and their use is legally restricted, whereas mass spectrometry requires sophisticated, costly, and sensitive instrumentation. To circumvent these difficulties, we adapted the nonradioactive iodothyronine deiodinase assay to determine DEHAL1 activity by a colorimetric readout, based on the Sandell-Kolthoff reaction. DEHAL1 was recombinantly expressed and used to optimize the assay in microtiter format. We applied the setup to scenarios of alternative substrate screening or search for compounds potentially acting as endocrine disrupting compounds, without identifying novel readily accepted substrates or inhibitors yet. Next, the assay was adapted to ex vivo material, and activity was reliably determined from rodent kidney and other tissues. Analyzing two mouse models of hyperthyroidism, we observed a decreased renal Dehal1 activity and mRNA expression. Our results show that this nonradioactive DEHAL1 assay is suited to screen for potential endocrine disrupters and to monitor endogenous Dehal1 expression. We harmonized the assay protocols to enable iodothyronine deiodinase and DEHAL1 activity measurements from the same samples. Hereby, a more complete view on iodine metabolism by these predominant deiodinating activities can be obtained from a given sample by a similar process flow.

Thr92Ala polymorphism of human type 2 deiodinase gene (hD2) affects the development of Graves' disease, treatment efficiency, and rate of remission.

Clinical symptoms vary in thyrotoxicosis, and severity of these depends on many factors. Over the last years, impact of genetic factors upon the development and clinical significance of thyrotoxic symptoms became evident. It is known that a production of T3 in various tissues is limited by deiodinase 2 (D2). Recent studies revealed that certain single nucleotide polymorphisms (including threonine (Thr) to alanine (Ala) replacement in D2 gene codon 92, D2 Thr92Ala) affect T3 levels in tissues and in serum. Individuals with Ala92Ala genotype have lower D2 activity in tissues, compared with that in individuals with other genotypes. In our study, we have assessed an association of D2 Thr92Ala polymorphism with (1) frequency of disease development, (2) severity of clinical symptoms of thyrotoxicosis, and (3) rate of remissions, in Graves' disease patients.

[Melatonin influence on free radical homeostasis in rat tissues at thyrotoxicosis].

Experimental thyrotoxicosis in rats is accompanied by the increase of serum alanine aminotransferase (AlA), aspartate aminotransferase (AsA), creatine kinase-MB (CK-MB) activities and content of primary products of lipid peroxidation--conjugated dienes--in liver, heart and blood. This suggests impairments in these organs accompanying free radical processes intensification. Administration of melatonin decreased AlA, AsA and CK-MB activities and CD level decreased. Thyrotoxicosis increased catalase activity in liver, heart and blood. Exogenous melatonin decreased specific activity ofcatalase in blood and in heart in comparison with animals subjected to hyperthyroidism. However, some increase of catalase specific activity (approximately 15%) was observed in liver. alpha-Tocopherol content, raising in rat tissues in thyrotoxicosis development conditions, decreased after melatonin treatment. Thus, exogenous melatonin is capable to reduce lipid peroxidation intensity at thyrotoxicosis and to act as an adoptogen, regulating free radical homeostasis.

The role of type 1 and type 2 5'-deiodinase in the pathophysiology of the 3,5,3'-triiodothyronine toxicosis of McCune-Albright syndrome.

CONTEXT: McCune-Albright syndrome (MAS) is caused by mutations in GNAS (most often R201C or R201H) leading to constitutive cAMP signaling and multiple endocrine dysfunctions, including morphological and functional thyroid involvement. OBJECTIVE: The objective of the study was to characterize the clinical and molecular features of the MAS-associated thyroid disease in a large cohort of patients. DESIGN: This was a retrospective analysis. SETTING: The study was conducted at the National Institutes of Health Clinical Center. PATIENTS: The study included 100 consecutive MAS patients. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURE: Functional and morphological evaluation of the thyroid was measured. Ex vivo experiments were performed on MAS thyroid samples to study the effects of the GNAS mutations on the 5'-deiodinases. Reconstitution experiments in HEK-293 cells were performed to study the effects of GNAS mutations on the type-2 5'-deiodinase. RESULTS: Fifty-four patients had abnormal thyroid ultrasound findings. This group, compared with patients without abnormal findings, had higher T(3) to T(4) ratio, indicating an elevated 5'-deiodinase activity. Thyroid samples from MAS subjects, compared with normal tissue, showed a significant increase in both type 1 (D1) and type 2 (D2) 5'-deiodinase activity (D1 control 5.9 +/- 4.5 vs. MAS 41.7 +/- 26.8 fmol/min.mg, P < 0.001; D2 control 28.3 +/- 13.8 vs. MAS 153.1 +/- 43.7 fmol/min.mg, P < 0.001). Compared with cells transfected with the wild-type R201 allele, the basal transcriptional activity of the D2 promoter was significantly increased in both mutants (C and H) (R 10733 +/- 2855, vs. C 18548 +/- 4514, vs. H 19032 +/- 4410 RLU +/- SD, P < 0.001). CONCLUSION: Thyroid pathology is a common occurrence in MAS. Consistent with the molecular etiology of the disease, the shift in T(3) to T(4) ratio is at least in part secondary to a cAMP-mediated intrathyroidal activation of D2 and to elevated D1 activity.

A cluster of thyrotoxicosis associated with consumption of a soy milk product.

OBJECTIVE: To determine whether a New Zealand cluster of thyrotoxicosis with low uptake on scintiscan was associated with soy milk consumption. METHOD: A case-control study was conducted, with controls matched by age, sex, and region. RESULTS: All cases (5/5) and one control (1/30) consumed soy milk before the index date. The cases all drank a brand of soy milk that had unexpectedly high levels of iodine. In a simple unmatched unadjusted analysis, the lower bound of the 95% confidence interval for the odds ratio was 19. CONCLUSION: This cluster was associated with consumption of a soy milk product. The thyrotoxicosis was probably due to iodine-induced thyrotoxicosis. IMPLICATIONS: This investigation raises issues about quality control in manufacturing processes and the monitoring of food products for their safety. It also raises issues about public and health professional awareness of the potential health effects from food additives in some processed foods.

Terminal intramuscular motor innervation and motor end-plates in thyrotoxic myopathy.

Thyrotoxic myopathy was induced in 64 mice. Examination of their muscles revealed excessive axonal branching and degenerative changes of preterminal axons. Moreover, the mean diameter of their end-plates decreased and the levels of end-plate cholinesterase appeared to be reduced. In 43 patients with thyrotoxic myopathy, increased axonal branching and degenerative changes of preterminal axons, similar to those in the experimental mice, were also seen. The possibility that excess thyroid hormone may interfere with axonal transport or neuromuscular interactions is discussed.

In vivo and in vitro sodium pump activity in subjects with thyrotoxic periodic paralysis.

OBJECTIVE: To examine whether sodium pump activity plays a part in the pathogenesis of thyrotoxic periodic paralysis. DESIGN: Measurement of platelet sodium-potassium ATPase and in vivo sodium pump activities in healthy subjects and thyrotoxic subjects with and without paralysis. SETTING: University hospital in Hong Kong. SUBJECTS: 21 healthy subjects, 23 untreated thyrotoxic subjects, 13 untreated men with periodic paralysis, seven treated thyrotoxic subjects, and six treated men with periodic paralysis. MAIN OUTCOME MEASURES: Platelet Na+, K(+)-ATPase activity and plasma rubidium concentration after oral loading. RESULTS: Median (range) platelet Na+, K(+)-ATPase activity in thyrotoxic subjects was 253 (169-821) mumol inorganic phosphate/h/g protein--significantly higher than that in healthy subjects (134 (81-180) mumol/h/g protein; p less than 0.001). Na+, K(+)-ATPase activity in those with periodic paralysis was 374 (195-1196) mumol/h/g protein, again significantly higher than that in healthy subjects (p less than 0.001) and that in other thyrotoxic subjects (p less than 0.01) despite similar degrees of hyperthyroidism. Activities in treated thyrotoxic subjects with and without periodic paralysis were 148 (110-234) and 131 (86-173) mumol/h/g protein respectively. Mean (95% confidence interval) plasma rubidium concentration five hours after oral administration in thyrotoxic subjects (7.0 (6.6 to 7.5) mumol/l) was significantly lower than in healthy subjects (10.2 (9.5 to 10.9) mumol/l; p less than 0.001) and higher than in those with periodic paralysis (6.0 (5.7 to 6.3) mumol/l; p less than 0.01). CONCLUSIONS: Sodium pump activity in untreated subjects with periodic paralysis is higher than in other thyrotoxic subjects, and this may be responsible for the hypokalaemia.

Evidence for acute release of thyroid peroxidase during subtotal thyroidectomy.

An immediate reduction of thyroglobulin autoantibodies during subtotal thyroidectomy of thyroglobulin antibody positive patients has previously been shown to indicate an acute release of thyroglobulin into the circulation peroperatively. The aim of the present study was to investigate whether thyroid peroxidase was also released by measuring anti-thyroid peroxidase antibodies by a quantitative and antigen specific method both pre- and postoperatively in patients positive for anti-thyroid peroxidase antibodies. Twelve anti-thyroid peroxidase positive patients (11 females, 1 male) referred for surgery of toxic goitre were studied. Median age was 43 years (range 24-64) and median goitre size 86 g (25-165). All patients had been pretreated with antithyroid drugs and were euthyroid at the time of operation. Anti-thyroid peroxidase was measured before operation, 1-8 h, 10 days, 1-3 months, and 12 months postoperatively by a commercial method (DYNO-test, Henning, Berlin). The median anti-thyroid peroxidase level before operation was 1048 kU/l (range 68-10 517 kU/l) and fell during operation to 0.63 (range 0.37-1.28) (p less than 0.01) of initial concentration without further decrease during the next 1-8 h. The comparative decrease in thyroglobulin antibodies was 0.19 (0-0.88). The anti-thyroid peroxidase level was increasing after 10 days, but did not reach initial level until between 3 and 12 months after surgery. However, in 3 of 10 patients anti-thyroid peroxidase had disappeared after 12 months, all of whom had low levels before operation, whereas anti-thyroid peroxidase was 2-4 times higher than preoperatively in 3 other patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The rate of tetanic relaxation is correlated with the density of calcium ATPase in the terminal cisternae of thyrotoxic skeletal muscle.

The density of calcium ATPase was measured in the terminal cisternae of extensor digitorum longus (EDL) and soleus muscles from normal and thyrotoxic rats. The experiments tested the hypothesis that the rate of relaxation of these muscles following contraction, at temperatures above 22 C, is correlated with the density of calcium ATPase in the sarcoplasmic reticular membrane. In soleus fibres there was a progressive increase in calcium ATPase density, measured with immuno-electronmicroscopic techniques, of more than two-fold after 3 weeks of daily injections with triiodothyronine (T3). There was a parallel decrease in the relaxation time (from 80% to 20% of peak tension) of the tetanus: the parameters were closely correlated (r = 0.998) during the 3-week period. The rate of relaxation of the twitch also doubled and was correlated with the increase in gold particle density at the end of the 3-week injection period. However, twitch relaxation slowed during the 1st week of T3 injection and was not correlated with gold particle density at that time. The changes in calcium ATPase density and relaxation times in EDL fibres were small and largely insignificant. In contrast to relaxation, an increase in the rate of rise of tension is soleus was complete after only 2 weeks of T3 injection. The results show that the relaxation of tetanic tension is closely correlated with the calcium uptake capacity of the sarcoplasmic reticulum and that thyroid hormone acts more rapidly on factors regulating the rate of rise of tension than on those regulating tension relaxation and the density of calcium ATPase in the terminal cisternae.

[An experimental study of the mechanisms of damage of the periodontal tissues in thyrotoxicosis]. / Eksperimental'noe izuchenie mekhanizmov povrezhdeniia tkanei parodonta pri tireotoksikoze.

Morphologic studies of the periodontal tissue in rats have revealed that destruction of the maxillodental system in excess of thyroid hormones is explained by disordered mineral metabolism, resorbed osseous tissue of the alveolar process, energy shifts, and developed inflammatory process. Introduction of physiologically-based amounts of calcium, iodine, and fluorine in the animals' ration at the early stages of their development reduced the degree of the alveolar process osseous tissue resorption.

Leukocyte alkaline phosphatase in hypothyroidism and hyperthyroidism. Response to initiation of thyroxine replacement therapy.

Leukocyte alkaline phosphatase (LAP) activity was determined in normal subjects, and in untreated, symptomatic patients with primary hypothyroidism or thyrotoxicosis. The means +/- 1 SD of (n) subjects were, respectively: 61.7 +/- 27.5 (16), 149.9 +/- 56.3 (9) and 96.9 +/- 27.7 (9). The mean LAP values of the hypothyroid and thyrotoxic groups were significantly different from that of the normal group (P less than .01). Values were above the normal range (20 to 120) in seven of the nine hypothyroid patients. LAP values were in the upper half of the normal range in eight of the nine thyrotoxic patients. In the two hypothyroid patients studied at 24-hour intervals, LAP activity was altered markedly within 48 hours of initiation of thyroxine therapy, 25 micrograms daily. In five hypothyroid patients followed for several months after initiating thyroxine replacement, LAP levels were essentially normal within 1 to 2 months. In the thyrotoxic patients, LAP values declined within the first month of medical management, but tended to remain within the normal range.

Comparison of ATPase activities and heavy chains of rabbit atrial and thyrotoxic ventricular myosin subfragment-1.

Subfragment-1 of rabbit atrial and thyrotoxic ventricular myosin (V1 isomyosin) has been prepared and purified by DEAE-cellulose column chromatography. Pyrophosphate-polyacrylamide gel electrophoretic patterns and column chromatographic profile of the atrial subfragment differ from those of thyrotoxic ventricular myosin subfragment-1. On the other hand, Ca2+, Mg2+ and actin-activated ATPase activities of these subfragments are identical. Comparison of the peptide mapping by limited proteolysis in the presence of sodium dodecyl sulfate of the heavy and the light subunits of these subfragments reveals that the patterns for the heavy chain peptides of these subfragments are substantially similar but their light chain peptide patterns differ. The results suggest that the enzymatic and structural similarities that have been recognized between these isoenzymes using intact myosin hold true for the myosin subfragment-1. The differences between these subfragments are due to the differences in the light chains associated with them.

The effect of thyroid dysfunction and fasting on placenta inner ring deiodinase activity in the rat.

The placenta contains iodothyronine 5-deiodinase activity (P5-Dase) that probably acts on iodothyronines in the fetal circulation to convert T4 to rT3 and T3 to 3,3'-T2. Since thyroid status and fasting have profound effects on iodothyronine deiodinases in other tissues, the present studies were performed to determine if these perturbations affected P5-Dase. Control and treated rats were mated and killed near term on the 20th day of gestation. P5-Dase was determined in placenta homogenates enriched with dithiothreitol by measuring the conversion of T4 to rT3. In four of five studies, P5-Dase was similar in dams that underwent thyroidectomy (Tx) on day 7 of gestation and sham Tx dams. P5-Dase was not altered in dams that were treated with methimazole (MMI) to induce maternal and fetal hypothyroidism. Treatment of dams with supraphysiological doses of T4, beginning on the seventh day of gestation, did not significantly affect P5-Dase. In three of four studies, P5-Dase was similar in fed dams to values in dams fasted for the last 5 days of pregnancy. Placenta iodothyronine 5'-deiodinase activity (P5'-Dase) was also measured in some studies. P5'-Dase was not decreased in Tx rats and was modestly decreased in MMI-treated rats. However, the effect of MMI was not reversed by the administration of supraphysiological doses of T4, Tx, MMI treatment, and fasting all decreased hepatic T4 5'-deiodinase activity in pregnant rats. These results strongly suggest that thyroid status and fasting do not alter P5-Dase activity.