Iodine fortification of vegetables improves human iodine nutrition: in vivo evidence for a new model of iodine prophylaxis.

BACKGROUND: Iodine deficiency is the result of insufficient intake of dietary iodine and as a consequence causes multiple adverse effects. About 2 billion individuals in the world are affected by iodine deficiency. It has been found that the most effective way to control iodine deficiency is through the universal salt iodization. However, salt iodization alone may not be sufficient to assure adequate iodine nutrition. In most industrialized countries, excess consumption of salt has become recognized as a health risk. Therefore, biofortification of vegetables with iodine offers an excellent opportunity to increase iodine intake. AIM AND METHODS: The aim of this study was to test the efficiency of a new model of iodine prophylaxis in a group of 50 healthy volunteers through the intake of vegetables (potatoes, cherry tomatoes, carrots, and green salad) fortified with iodine. Each serving of vegetables consisted of 100 g of potatoes, carrots, tomatoes, or salad containing 45 mg of iodine (30% of the Recommended Daily Allowance), and the volunteers consumed a single serving of vegetables, as preferred, each day for 2 weeks. Urinary iodine (UI) excretion was measured before and after intake of vegetables. RESULTS: The UI concentration measured in volunteers before the intake of vegetables was 98.3 mg/L (basal value), increasing to 117.5 mg/L during the intake of vegetables. Seven days after the discontinuation of vegetable intake, UI was 85 mg/L. UI concentration increment was 19.6% compared with the basal value; therefore, the difference was statistically significant (P = .035). CONCLUSIONS: Biofortification of vegetables with iodine provides a mild but significative increase in UI concentration and, together with the habitual use of iodized salt, may contribute to improve the iodine nutritional status of the population without risks of iodine excess.

Identification and functional analysis of novel dual oxidase 2 (DUOX2) mutations in children with congenital or subclinical hypothyroidism.

CONTEXT: Congenital hypothyroidism (CH) associated with goiter or a gland of normal size has been linked to dual oxidase 2 (DUOX2) mutations in the presence of iodide organification defect. OBJECTIVE: Thirty unrelated children with CH or subclinical hypothyroidism (SH) identified during infancy with a eutopic thyroid gland, coming from our Screening Centre for CH or referred from other regions of Italy, were studied with the perchlorate discharge test to identify organification defects. Eleven children with iodide organification defect were considered for the genetic analysis of TPO, DUOX2, and dual oxidase maturation factor 2 (DUOXA2) genes. PATIENTS: Eight children with CH and three with SH and eutopic thyroid gland were included in the study. After discontinuation of therapy, a partial or complete organification defect was shown after ¹²³I scintigraphy and perchlorate test. METHODS: TPO, DUOX2, and DUOXA2 genes were analyzed, and functional activity of DUOX2 variants was studied in HeLa cells. RESULTS: Sequencing of the DUOX2 gene revealed a deletion S965fsX994 in three children; two were euthyroid after 1 month of L-T4 discontinuation but developed SH after 5 and 18 months, respectively, whereas the other child had SH. One child with SH showed H678R, R701Q, and P982A substitutions, and another child with SH showed only the P982A. One child with SH showed the Y1150C mutation, and another euthyroid child showed the A728T mutation. Functional studies confirmed that S965fsX994, Y1150C, and A728T mutations were responsible for the defect in H2O2 production, whereas H678R, R701Q, and P982A did not alter H2O2 production in vitro. CONCLUSIONS: Genetic analysis of the DUOX2 gene was performed in 11 children with organification defect. Two new mutations (Y1150C and A728T) and the deletion S965FsX994 were responsible for the deficit in the organification process and the phenotypes. Three polymorphisms (H678R, P982A, and R701Q) were identified.

Evidence for protein and mRNA TSHr expression in fibroblasts from patients with thyroid-associated ophthalmopathy (TAO) after adipocytic differentiation.

OBJECTIVE: Thyroid-associated ophthalmopathy (TAO) is a chronic autoimmune disorder characterized by an increased volume of adipose/connective tissue in the human orbit. DESIGN: The aim of this study was to investigate the thyrotropin receptor (TSHr) expression in orbital fibroblasts from TAO patients undergoing adipocytic differentiation. METHODS: Retro-ocular tissue and skin were obtained from five patients undergoing orbital decompression surgery for TAO and placed in culture. Proliferating fibroblasts were subjected to adipocytic differentiation for 10 days. Total RNA was isolated from fibroblasts and was reverse transcribed. TSHr mRNA levels were determined by real-time PCR. cAMP was determined by radioimmunoassay (RIA) after fibroblast incubation with the substances to test. RESULTS: Orbital differentiated fibroblasts became rounded and acquired lipid droplets. The amount of TSHr mRNA in these fibroblasts was higher than fibroblasts not subjected to adipocytic differentiation. Immunocytochemical analysis showed TSHr protein in differentiated orbital fibroblasts. Differentiated orbital fibroblasts stimulated with bovine (b) TSH showed a cAMP production greater than that in paired undifferentiated cultures. A specific thyroid-inhibiting antibody (TBAb) inhibited cAMP production after bTSH challenge, and a thyroid-stimulating antibody (TSAb) stimulated cAMP production in differentiated fibroblasts. CONCLUSIONS: We suggest that orbital fibroblasts subjected to adipocytic differentiation increase TSHr expression that responds specifically to bTSH and TSAb stimulation, and to TBAb inhibition.

Gonadotrophin receptor blocking antibodies measured by the use of cell lines stably expressing human gonadotrophin receptors are not detectable in women with 46,XX premature ovarian failure.

BACKGROUND: Premature ovarian failure (POF) is defined by cessation of ovarian function after puberty and before the age of 40. The syndrome is characterized by amenorrhoea, oestrogen deficiency and elevated levels of gonadotrophins. Autoimmunity has been proposed as a mechanism for some cases of destruction or malfunction of ovarian follicles. POF is often associated with type I and type II polyglandular autoimmune syndromes. It has also been postulated that receptors such as the LH and FSH receptors might become targets for blocking antibodies and such antibodies could be a cause of ovarian failure. PATIENTS AND METHODS: Sixty-nine patients with POF isolated or associated with other endocrine autoimmune diseases (autoimmune thyroid diseases, Addison's disease, type 1 diabetes mellitus, multiple sclerosis, myasthenia gravis) were studied. All the patients had secondary amenorrhoea. The patient group had a median age of 33.1 years (range 15-57). Ovarian failure had been diagnosed at a median age of 29 years (range 15-39). The median time since diagnosis was almost 1 year but in six patients gonadal insufficiency had appeared 10-30 years earlier. All had a normal chromosomal karyotype (46, XX). Patients with POF were characterized by duration of amenorrhoea > 1 year, with elevated FSH and LH levels and undetectable or low oestrogen levels. Cell lines stably expressing recombinant human LH (CHO-LHr) and FSH (CHO-FSHr) receptors were prepared and used to search for antibodies able to inhibit LH- or FSH-stimulated cAMP production. Immunoglobulins extracted from sera of patients with POF were incubated with CHO-LHr and CHO-FSHr in the presence of human recombinant CG and FSH, respectively. RESULTS AND CONCLUSIONS: None of the immunoglobulin G (IgG) preparations from patients with POF was able to inhibit the activity of the FSH- and CG-stimulated cAMP production.

The sodium-iodide symporter protein is always present at a low expression and confined to the cell membrane in nonfunctioning nonadenomatous nodules of toxic nodular goitre.

OBJECTIVE: The sodium-iodide (Na(+)/I(-)) symporter (NIS) is known to be overexpressed in toxic adenomas and in about half of benign solitary nonfunctioning adenomas of the thyroid. In nonfunctioning adenomas, however, the protein is localized mainly in the cytoplasm and fails to reach the basolateral membrane of the follicular cell where it is found in normal thyroid tissue and in hyperfunctioning adenomas. Our aim was to study both the level of expression and the cell localization of NIS in nonfunctioning nonadenomatous nodules of toxic nodular goitre. DESIGN AND PATIENTS: Tissue specimens from nine patients who were submitted to surgery for toxic or functionally autonomous goitre were studied. Tissues from 12 patients who underwent lobectomy for a toxic thyroid adenoma were used as controls. MEASUREMENTS: Tissue sections embedded in paraffin were stained with haematoxylin-eosin for histological evaluation and for immunohistochemistry using a monoclonal antibody that recognized human (h) NIS. RESULTS: Functioning and nonfunctioning nodules scattered in multinodular goitres consisted of unencapsulated micro/macrofollicular aggregates. All toxic adenomas were characterized by a micro/macrofollicular pattern of growth and histological examination showed that they were surrounded by a capsule. Like the 12 toxic adenomas, all the 14 functioning hyperplastic nodules and two adenomas of toxic multinodular goitre showed a high level of hNIS protein expression with respect to the normal collateral parenchyma and, in all cases, the protein was confined to the cell membrane. CONCLUSIONS: Contrary to what was observed for solitary nonfunctioning thyroid adenomas, all the 19 nonfunctioning hyperplastic nodules showed a very low hNIS and this was always confined to the cell membrane. These data suggest that the mechanisms leading to loss of iodide uptake in nonfunctioning hyperplastic nodules of toxic multinodular goitre are different from those that act on solitary nonfunctioning follicular adenomas.

TSH receptor antibodies do not alter the function of gonadotropin receptors stably expressed in eukaryotic cells.

OBJECTIVE: TSH receptor (TSHr) mediates the activating action of TSH on the thyroid gland resulting in the growth and proliferation of thyrocytes and thyroid hormone production. TSHr is a major autoantigen in Graves' disease (GD) and is the target for TSHr antibodies. In GD, thyroid-stimulating antibodies (TSAb) are competitive agonists of TSH. In atrophic thyroiditis (AT), thyroid-stimulating blocking antibodies (TSHBAb) are TSH antagonists. The TSHr together with the LH receptor (LHr) and FSH receptor (FSHr) are G-protein-coupled receptors with considerable amino acid homologies in the extracellular domain. We studied the cross-reactivity of the antibodies measured in sera from patients with GD or AT on the LHr and FSHr function. METHODS: We tested the activity of TSAb and TSHBAb in cell lines expressing the LHr and the FSHr. To this purpose a pSVL-FSHr construct was transfected in CHO cells and one clone was used. RESULTS: Twenty-eight sera from patients with GD and four from patients with AT, known to contain TSHr antibodies measured with a radioreceptor assay, were selected. TSAb and TSHBAb activities were measured in CHO cells expressing the TSHr (CHO-TSHr). TSAb and TSHBAb were then tested with the cell lines expressing the LHr and the FSHr for their ability to elicit cAMP accumulation or inhibit FSH/LH-induced cAMP production. None of the TSAb identified was able to stimulate cAMP increase in CHO-LHr or CHO-FSHr. Similarly, none of the TSHBAb was able to block the cAMP response induced by FSH or LH in the respective cell lines. CONCLUSIONS: Our results confirm the notion of the organ-specific nature of the TSHr antibodies.

Congenital hypothyroidism due to a new deletion in the sodium/iodide symporter protein.

OBJECTIVE: Iodide transport defect (ITD) is a rare disorder characterised by an inability of the thyroid to maintain an iodide gradient across the basolateral membrane of thyroid follicular cells, that often results in congenital hypothyroidism. When present the defect is also found in the salivary glands and gastric mucosa and it has been shown to arise from abnormalities of the sodium/iodide symporter (NIS). PATIENT: We describe a woman with hypothyroidism identified at the 3rd month of life. The diagnosis of ITD was suspected because of nodular goitre, and little if any iodide uptake by the thyroid and salivary glands. Treatment with iodide partially corrected the hypothyroidism; however, long-term substitution therapy with L-thyroxine was started. MEASUREMENTS: Thyroid radioiodide uptake was only 1.4% and 0.3% at 1 and 24 h after the administration of recombinant human TSH. The saliva to plasma I- ratio was 1.1 indicating that the inability of the thyroid gland to concentrate I- was also present in the salivary glands. RESULTS: Analysis of the patient's NIS gene revealed a 15 nucleotide (nt) deletion of the coding sequence (nt 1314 through nt 1328) and the insertion of 15 nt duplicating the first 15 nt of the adjacent intron. The patient was homozygous for this insertion/deletion, while both consanguineous parents were heterozygous. This deletion predicts the production of a protein lacking the five terminal amino acids of exon XI (439-443) which are located in the 6th intracellular loop. COS-7 cells transfected with a vector expressing the mutant del-(439-443) NIS failed to concentrate iodide, suggesting that the mutation was the direct cause of the ITD in this patient. CONCLUSION: In conclusion we describe the first Italian case of congenital hypothyroidism due to a new deletion in the NIS gene.

Benign nonfunctioning thyroid adenomas are characterized by a defective targeting to cell membrane or a reduced expression of the sodium iodide symporter protein.

Nodular thyroid disease is the most common endocrine disorder. Nonfunctioning thyroid nodules are identified by their low radioiodide uptake compared with the normal extranodular tissue, which, at thyroid scintiscan, produces the typical picture of a cold thyroid nodule. Previous in vitro studies demonstrated that the majority of nonfunctioning thyroid nodules have a specific defect in iodide transport that accounts for their failure to accumulate radioactive iodide in vivo. A defect in the expression or structure of the sodium iodide symporter (NIS) gene has been hypothesized as a possible cause of the impaired iodide trapping in nonfunctioning thyroid nodules. We studied 22 patients who were submitted to surgery for a solitary nonfunctioning thyroid nodule that originated in an otherwise normal gland. Thyroid scintigraphy was performed at 1, 2, 3, 4, 6, and 24 h after the oral administration of a tracer dose of 131I (iodine). All patients showed absence of 131I uptake in the nodule, with normal uptake in the extranodular tissue and in the contralateral thyroid lobe. Eight patients with toxic adenomas who underwent lobectomy were also included in the study. We first studied the expression of human NIS (hNIS) protein by immunohistochemistry in paraffin-embedded tissue sections using a specific anti-hNIS monoclonal antibody. Subsequently, we searched for somatic mutations of hNIS gene in nonfunctioning thyroid nodules. The level of hNIS expression was determined in both the nodules and the normal tissue from the same thyroid gland. In all functioning thyroid nodules (toxic adenomas), a high expression of hNIS protein was detected with respect to normal surrounding tissue. Similar to the normal thyroid tissue, follicular cells of toxic thyroid adenomas showed an exclusive expression of hNIS protein at the cell membrane. Fifty-four percent of benign nonfunctioning thyroid nodules overexpressed hNIS protein compared with the normal surrounding tissue, but in these nodules the hNIS protein failed to target the cell membrane, being mostly localized inside the cytoplasm. hNIS protein was not detected by immunohistochemistry in 46% of nonfunctioning nodules, whereas it was expressed in the surrounding unaffected thyroid tissue. Direct sequencing of the hNIS gene in all of the nonfunctioning nodules did not reveal major genetic alterations. A silent polymorphism (GCC/GCG codon 544, exon 13) was found in one nodule. In conclusion, the results obtained in this study show that two mechanisms contribute to the reduced radioiodide uptake typical of benign nonfunctioning thyroid nodules: 1) reduced expression of the hNIS protein, and 2) defective targeting of hNIS to the cell membrane.