Deiodination Of Iodotyrosines Research Articles

Iodotyrosine dehalogenase 1 (DEHAL1) is a transmembrane protein involved in the recycling of iodide close to the thyroglobulin iodination site.

In the thyroid, iodotyrosine dehalogenase acts on the mono and diiodotyrosines released during the hydrolysis of thyroglobulin to liberate iodide, which can then reenter the hormone-producing pathways. It has been reported that the deiodination of iodotyrosines occurs predominantly in the microsomes and is mediated by NADPH. Recently, two cDNAs, 7401- and 7513-base pairs long that encode proteins with a conserved nitroreductase domain were published in GenBank as iodotyrosine dehalogenase 1 (DEHAL1) and iodotyrosine dehalogenase 1B (DEHAL1B), respectively. We report here our investigation of the localization and activity of one of these isoforms, DEHAL1. DEHAL1 mRNA is highly expressed in the thyroid, is up-regulated by cAMP, and encodes a transmembrane protein that efficiently catalyzes the NADPH-dependent deiodination of mono (L-MIT) and diiodotyrosine (L-DIT), with greater activity vs. L-MIT. Iodotyrosine deiodinase was active in HEK293 cells transfected by DEHAL1 cDNA, but not in CHO cells. A fraction of DEHAL1 protein is exposed to the cell surface, as indicated by biotinylation experiments. Immunohistochemistry studies showed that DEHAL1 proteins accumulate at the apical pole of thyrocytes. Taken together, these findings indicate that the deiodination reaction occurs at the apical pole of the thyrocyte and is involved in a rapid iodide recycling process at and/or close to the organification site.

Studies on the mechanism of inhibition of thyroid function by lithium.

The effect of dietary lithium on the rate of thyroglobulin degradation was investigated using both in vivo and in vitro experimental models. The in vivo experiments utilized a modification of the double isotopic method of estimation of protein turnover (Arias, I.M., Doyle, D and Schminke, R.T. (1969) J. Biol. Chem. 244, 3303–3315) injecting isotopes of iodine, 131 I and 125 I, to label thyroglobulin 27 h and 3 h respectively before killing. Faster rates of thyroglobulin hydrolysis were associated with lower activity of 131 I as compared to 125 I and thus resulted in higher 125 I/ 131 I isotopic ratios in thyroglobulin. The isotopic ratio in the 1000 × g supernatant fraction of thyroid gland homogenate closely approximated the isotopic ratio in the isolated thyroglobulin and was used as an estimate of the rate of thyroglobulin degradation. Treatment with lithium had no direct effect on the rate of thyroglobulin degradation. The only exception was observed in states of extreme iodine deficiency, presumably through suppression of thyrotropin release by the pituitary. Experiments in vitro were carried out by labeling of the thyroglobulin pool in vivo with an injection of 131 I and subsequent culture of the isolated glands for 6 h in vitro under conditions where deiodination of iodotyrosines was prevented (Bagchi, N., Brown, T.R., Shivers, B. and Mack, R.E. (1977) Endocrinology 100, 1002–1008). The rate of thyroglobulin degradation, as measured by the percent 131 I released as iodotyrosines and iodothyronines, was not altered by lithium administration. However, lithium-treated animals demonstrated a marked decrease in the rate of release of 131 I-iodothyronines. The effect of lithium on thyroglobulin iodination was investigated in parallel in vivo experiments. Lithium administration resulted in a decreased content of iodothyronines and a lower moniodotyrosine/diiodotyrosine ratio. These studies suggest that lithium acts primarily by inhibition of the coupling of iodotyrosines and has no direct effect on thyroglobulin degradation. Hydrolysis of such iodothyronine-poor thyroglobulin would account for the observed decreases of hormonal release from the gland.

Congenital Defect in Iodothyronine Synthesis. Clinical Aspects of Iodine Metabolism in Goats with Congenital Goitre and Hypothyroidism

SUMMARY Iodine metabolism was studied in seven normal goats and six goats with congenital hypothyroidism and goitre. The goitrous goats were greatly retarded in growth, their skin was thick and scaly, and they had a sparse haircoat. Thyroidal turnover of radioiodine was markedly increased and was not influenced by the administration of KCNS or methimazole. There was no apparent defect in deiodination of iodotyrosines. Column chromatography of plasma revealed that most of the thyroidal radioiodine was released into the circulation as an iodopolypeptide. No free MIT or DIT could be detected in the plasma or urine. These findings suggest that the goitrous goats had a defect, comparable to those found in man and sheep, in thyroglobulin synthesis, with the release of abnormal iodinated proteins into the circulation.

Effect of inorganic iodide on thyroglobulin hydrolysis in cultured thyroid glands.

The process of thyroglobulin hydrolysis in mouse thyroid glands labelled in vitro was studied from 2-24 h after they had been maintained in tissue culture. The culture medium was supplemented with mononitrotyrosine to prevent deiodination of iodotyrosines. Hydrolysis of labelled thyroglobulin, under these conditions, led to the release of labelled iodotyrosines and iodothyronines. The rate of formation of these compounds was measured as an index of thyroglobulin hydrolysis (TH). TH was markedly stimulated by TSH. NaI inhibited TSH stimulation of TH at a concentration of 10(-5)M or greater. NaI, at similar concentrations,also markedly diminished or abolished the incorporation of 131I into thyroidal proteins from radioiodide-supplemented media. The addition of various inhibitors of iodination effectively blocked the effect of iodide on TH. In experiments where radioimmunossay was used to measure medium hormone concentrations, the release of unlabelled thyroxine (T4) and triiodothyronine (T3) induced by TSH was found to be significantly decreased in the presence of 10(-4)M NaI. These studies demonstrate that iodide inhibits the hydrolysis of thyroglobulin at or near concentrations which also inhibit iodination of thyroidal proteins. The present data suggest that formation of an iodinated compound is necessary for the effect of iodide. In addition, these studies demonstrate the utility of this in vitro system for the investigation of thyroid physiology.

Evidence for a secretion of thyroxine by isolated hog thyroid cells

Isolated thyroid cells prepared from hog thyroid glands by tryptic dispersion were incubated with 131I − for 1–6 h. Free [ 131I]thyroxine was identified in the incubation medium by three chromatographic methods. Neither [ 131I]iodotyurosines nor [ 131I]triiodothyronine were detected. The [ 131I]thyroxine released in the medium by 100 μl of cells (packed cell volume) after a 6-h incubation period amounted to 1.16% (S.E. = ± 0.39) of the total radioactivity. The medium [ 131I]thyroxine represented 15–25% of the total [ 131I]thyroxine synthesized during the 6 h of incubation. Thyrotropin, 1–60 munits/ml, increased the medium [ 131I]thyroxine content 2–4 fold. Dibutyryl cyclic AMP mimicked the effect of thyrotropin. The amount of medium [ 131I]thyroxine was strictly related to the amount of incubated cells but was independent of the volume of the incubation medium. When prelabeled cells were incubated in the presence of methimazole the increase in medium [ 131I]thyroxine was quantitatively related to a decrease in the intracellular [ 131I]thyroxine. Addition of dinitrotyrosine, an inhibitor of the deiodinase activity, induced the release of iodotyrosines in the incubation medium. That the incubation supernatant of isolated thyroid cells did contain free thyroxine but no iodotyrosines suggests that the normal mechanisms of proteolysis of thyroglobulin and deiodination of iodotyrosines inside the cells are preserved. From these data, it was concluded that the thyroxine release by isolated cells represents a real secretion.

EFFECT OF LITHIUM ON DEIODINATING ACTIVITY OF VARIOUS RAT TISSUES IN VITRO

ABSTRACT The effect of lithium chloride (LiCl) on the deiodination of iodotyrosines, on the degradation of 125I-L-thyroxine (125I-L-T4) in vitro and on the disappearance of exogenous 125I-L4 and 125I-rat-TSH in vivo was studied in rats. Iodotyrosine deiodination was studied in vitro with three techniques. The whole thyroid lobes were not satisfactory as substrate. When a diluted mixture of prelabelled iodo-amino acids was used as substrate, thyroid homogenates deiodinated iodotyrosines. The reaction was inhibited by boiling and by 3,5-dinitro-L-tyrosine (DNT), but LiCl (2 × 10−2 m) had no effect. When 125I-3-iodo-L-tyrosine (125I-L-MIT) served as substrate, increasing concentrations of thyroid homogenates showed an increasing deicdinating activity, which was stimulated by NADP (1.5 × 10−4 m). Inhibitors of dehalogenase DNT (10−4 and 10 −3 m) and menandione (10−4 m) inhibited deiodination, but LiCl (5×10−3 − 0.1 m) was again without effect. The degradation of 125I-L-T4 by liver and kidney homogenates was inhibited by LiCl (5 × 10−3 − 0.1 m). The disappearance of 125I-L-T4 was studied in rats treated with LiCl for 1 – 4 or 60 – 64 days in vivo. The half-lives were as follows: at 1 –4 days, the control rats 15.9 ± 1.3 h and the LiCl treated rats 19.1 ± 2.1 h (P < 0.05) and at 60 – 64 days 11.2 ± 2.0 h and 66.8 ± 12.3 h (P < respectively. The prolonged half-life in the LiCl treated rats was not due to the decreased excretion of radioactivity in the urine or faeces. The biological half-life of 125I-rat-TSH (11.4 ± 3.2 min) was not modified by LiCl treatment for 5 days. It can be concluded that the antithyroid effect of LiCl neither originates from the inhibition of iodotyrosine deiodination nor from the change in the half-life of TSH. The half-life of thyroxine is prolonged by LiCl, an effect which is perhaps due to the decreased degradation of thyroxine by tissues.

Influence of perchlorate on the secretion on non-thyroxine iodine by the normal human thyroid gland.

Abstract. Several authors have postulated that endogenous iodide produced by the deiodination of iodotyrosines in the thyroid feeds into a different thyroidal iodide compartment than transported iodide which enters the gland from outside. One argument for the existence of two separate iodide compartments is the observation that under certain experimental conditions perchlorate completely discharges transported iodide from the thyroid, while it has no such effect on endogenous iodide. This latter observation however has not been confirmed by all studies and remained controversial. – We therefore reinvestigated the effect of perchlorate on the secretion of endogenous iodide by a new, sensitive method. Five normal volunteers received tracer amounts of iodide‐125I p.o. and 11 days later thyroxine‐131I I.V. Two days later the following serial measurements were started: serum protein‐bound labelled iodine (PB125I, PB131), serum total thyroxine and urinary excretion of 125I, 127I and 131I. – In the control period the non‐thyroxine iodine secretion calculated from the above measurements was 40 μg/day. Under perchlorate 200 mg three times daily this value rose significantly to 66 μg/day. Non‐thyroxine iodine comprises the secreted triiodothyronine plus the secreted endogenous iodide. Assuming that the former value remained constant, our data show that perchlorate indeed discharges part, though not all, of the endogenous iodide. These data do not rule out a second iodide compartment, but they are also compatible with a simple one compartment model.

Secretion of thyroxine and non-thyroxine iodine by the normal human thyroid gland. Influence of carbimazole and pharmacological doses of iodide.

Abstract. In order to measure the effect of antithyroid agents on thyroxine and non‐thyroxine iodine secretion by the normal human thyroid gland, 12 normal persons were given tracer amounts of iodide‐125I. 7 to 11 days later thyroxine‐131I was injected I.V. Two days later the following serial measurements were started: Serum protein‐bound labeled iodine (PB125 PB131I), serum total thyroxine, and excretion of 125I and 131I in urine.–In the control period (no drugs) the secretion rate for thyroxine was 83 μg/day. Carbimazole (15 mg three times daily) for 6 days did not affect thyroxine secretion despite a complete block of iodine uptake by the thyroid. Non‐thyroxine iodine secretion was 59 μg/day in the control period and rose to 183 μg under carbimazole.–Non‐thyroxine iodine includes triiodothyronine and any non‐hormonal iodine, such as iodide derived from the deiodination of iodotyrosines, secreted by the gland. Based on recently published estimates it was assumed that the triiodothyronine contribution was not more than 24 μg. This left a considerable efflux of non‐hormonal iodine even in the control period. Most of the non‐hormonal iodine produced, however, was recycled into thyroglobulin without leaving the gland. After blocking reutilization by carbimazole large amounts of non‐hormonal iodine were secreted by the gland. High doses of iodide (30 mg three times daily) in combination with carbimazole led to a rapid 45% decline in thyroxine secretion. Since a complete block of hormone synthesis by carbimazole alone had no such effect, high iodide doses must act at a different site, presumably by slowing down the release of hormone from preformed thyroglobulin stores. The findings indicate that carbimazole enhances the sensitivity of the normal thyroid gland toward pharmacological doses of iodide.–High doses of iodide together with carbimazole reduced the secretion of non‐hormonal iodine (which can be taken as an index of thyroglobulin hydrolysis) by only 31%. Moreover some subjects showed no reduction at all, despite of concomitant fall in hormone secretion. The lack of correlation between the decreases of thyroxine and of non‐hormonal iodine secretion does not support the generally held view that high doses of iodide block hormone release by interfering with the hydrolysis of thyroglobulin.

Decrease in the thyroid internal iodide pool induced by 3-nitro-L-tyrosine (MNT), an inhibitor of iodotyrosine deiodination.

Studies have been performed to evaluate further the postulate that the thyroid contains as a distinct functional entity an “internal” or “second” thyroidal iodide pool, arising from intrathyroidal deiodinations, in addition to an iodide pool, representing iodide transported in the thyroid from the extracellular fluid. Thyroidal iodine metabolism was determined after administration of nitro—L—tyrosine (MNT), a potent inhibitor of the deiodination of iodotyrosines. MNT, given acutely to rats, resulted in a significant decrease in the measured iodide content of the internal iodide pool and the internal thyroid/serum iodide concentration ratio, without changing significantly the thyroidal organic iodine content, the iodide content of the external iodide pool or the external thyroid/serum iodide concentration ratio. These results provide additional evidence that the measured internal iodide pool does not represent iodide derived artifactually during analysis, but is indeed a distinct physiological e...

Failure of dinitrotyrosine to affect the TSH-induced acute depression of thyroidal iodide transport.

Dinitrotyrosine (DNT), a potent inhibitor of thyroid iodotyrosine deiodinase, failed to affect the acute TSH-induced depression of the thyroid-.serum radioiodide concentration [T/S(I~)]. This finding lends support to the concept that the acute TSH-induced depression of T/S(I~) is not caused by expansion of the intrathyroidal iodide pool consequent upon increased proteolysis of thyroglobulin and deiodination of free iodotyrosines following TSH administration. (Endocrinology 88:1277,1971) T (TSH)-induced release of iodide from thyroids was reported by Becker et al. (1) in 1953 and other groups (2). Halmi et al. (3) described a biphasic effect of TSH on thyroid iodide concentration in the rat with an early depression of the thyroid: serum radioiodide ratio [T/S(I~)], 4-5 hr after injection of TSH, followed by a later rise which exceeded the pre-TSH levels. The early depression was explained by them as due to an increased exit rate for iodide (4). Rosenberg (2) and Wolff (5) considered TSH-induced hydrolysis of thyroglobulin with subsequent deiodination of iodotyrosines and expansion of the thyroid iodide pool as another possible cause. However, studies (3) in iodine-deprived rats whose thyroid glands would contain relatively small amounts of iodotyrosines showed a TSHinduced early depression of the T/S(I~), similar to that of animals not deprived of iodine. In the present experiments, the problem has been explored further by inhibiting deiodination of free intrathyroidal iodotyrosines by administration of dinitrotyrosine (DNT). Materials and Methods Experiments with in situ perfusion. To demonstrate the inhibitory effect of DNT administered in vivo on intrathyroidal deiodination of iodotyrosines, male Sprague-Dawley rats weighing 200-300 g were fed Purina laboratory chow or a low iodine diet (approximately 30 Mg I/kg, General BioReceived February 26, 1970. This investigation was supported by Grants AM-01447 and AM-05065 from the National Institute of Arthritis and Metabolic Diseases, NIH. The work was completed with support from Grant 36X0161, Medical Research VA, 1970. 1 Trainee in Endocrinology of the National Institute of Arthritis and Metabolic Diseases. Present address: Veterans Administration Hospital, Jefferson Barracks Division, St. Louis, Mo. 63125. chemicals, Inc., Chagrin Falls, Ohio). They were injected with carrier-free I~ 16—24 hr and with DNT 105-120 min before exsanguination. Sufficient I was injected ip to obtain a thyroid content of 30-60 /uCi at the time of perfusion. DNT was prepared as described below and injected sc in the doses shown in Table 1. Immediately after sacrifice, the thyroid glands were perfused in situ by the technique developed in this laboratory (6). The entering perfusate contained bovine thyrotropin (TSH) (a gift of the National Institute of Health, lot NIH-B4), 1 U/100 ml, to maintain thyroid secretion. The total radioactivity secreted per min and the relative distribution of labeled compounds did not appreciably change over the first 30 min of perfusion when TSH was added to the perfusate (6). The perfusion lasted 30-45 min. Two or 3 thyroid effluent collections were made. Only the last one is presented in Table 1. Thyroid effluent plasma, pancreatin digests of the glands removed after perfusion, and the plasma obtained during exsanguination before perfusion were analyzed by paper chromatography in butanol-ethanol-ammonia (BEA) or butanol-acetic acid-water (BAW) systems to determine the relative quantitative distribution of labeled iodoamino acids and iodide (6). Origin material was not included in the calculation of the relative distribution. The degree of inhibition of iodotyrosine deiodination was estimated by comparison of the per cent of labeled MIT and DIT in the effluent with that in the thyroid digest. The mean effluent percentage iodotyrosines of all DNT-treated rats in Table 1 (57.3+1.8 SEM) was divided by that of the digest (68.9+2.1 SEM), which was 83%. It was assumed that the inhibition is complete when the percentage iodotyrosines secreted equals that of the digested thyroglobulin. Greer and Grimm (7) achieved almost complete inhibition by this criterion when they perfused prelabeled thyroids with blood containing 10~ and 10~M DNT. Experiments involving T/S(I~) ratios. Male Sprague-Dawley rats weighing 120-150 g were fed Purina chow and given tap water ad lib. TSH was obtained from the Armour Pharmaceutical Co.,

THE POSSIBLE OCCURRENCE OF TWO INBORN ERRORS OF IODINE METABOLISM IN ONE PATIENT.

ABSTRACT The occurrence of two separate inborn errors of iodine metabolism in one patient has been suggested by some authors but doubted by others. The combination of two other mechanisms may explain most of these cases: one defect may be conditioned by another, or the defects may have a common underlying cause. A seemingly impeded deiodination of iodotyrosines, found together with one of the other defects in a hypothyroid patient, may be an example of the first mechanism. The second mechanism might be involved when the primary abnormality is located in the biogenesis of thyroglobulin. In the absence of normal thyroglobulin, three successive reactions could secondarily be influenced: the iodination, the »coupling« and the proteolysis. Thus, when two or more of these reactions are found to be (partially) blocked, defective thyroglobulin biogenesis should be considered as a possibility. A case is presented in which such a defect was at first suggested by the simultaneous occurrence of circulating iodinated protein-like compounds (defective proteolysis) and the secretion of large amounts of iodide by the thyroid (hampered coupling), and was subsequently confirmed by the electrophoretic analysis of a thyroid extract which showed an absence of normal thyroglobulin. Furthermore, additional evidence is presented on a similar patient described in a previous paper. Finally, a survey is given of sixteen other patients described in the literature in whom the existence of defective thyroglobulin biogenesis was either suggested by the occurrence of two seemingly separate defects or confirmed by the absence of normal thyroglobulin.

Metabolism of monoiodotyrosine, diiodotyrosine, triiodothyronine and thyroxine by L-amino acid oxidase.

Thyroxine, triiodothyronine, diiodotyrosine and monoiodotyrosine, labelled with I131, were incubated with L-amino acid oxidase of cobra venom, and their metabolites studied by paper chromatography and radioautography. Under these conditions, the iodinated tyrosines were converted to the corresponding alpha-keto acid and acetic acid, inorganic iodide, and a number of unknown compounds. The deiodination of iodotyrosines by L-amino acid oxidase occurred after their corresponding alpha keto acids were formed by deamination of theamino acids. Triiodothyronine and thhyroxine were converted to their respective acetic acid derivatives, but not to inorganic iodide. The rate of degradation of these aromatic iodinated compounds by L-amino acid oxidase was related to the number of iodine atoms in the phenyl or diphenyl ether radical; i.e. monoiodotyrosine→diiodotyrosine→triiodothyronine→thyroxine.