Mass spectrometry in the diagnosis of thyroid disease and in the study of thyroid hormone metabolism

The importance of thyroid hormones in the regulation of development, growth, and energy metabolism is well known. Over the last decades, mass spectrometry has been extensively used to investigate thyroid hormone metabolism and to discover and characterize new molecules involved in thyroid hormones production, such as thyrotropin-releasing hormone. In the earlier period, the quantification methods, usually based on gas chromatography-mass spectrometry, were complicated and time consuming. They were mainly focused on basic research, and were not suitable for clinical diagnostics on a routine basis. The development of the modern mass spectrometers, mainly coupled to liquid chromatography, enabled simpler sample preparation procedures, and the accurate quantification of thyroid hormones, of their precursors, and of their metabolites in biological fluids, tissues, and cells became feasible. Nowadays, molecules of physiological and pathological interest can be assayed also for diagnostic purposes on a routine basis, and mass spectrometry is slowly entering the clinical laboratory. This review takes stock of the advancements in the field of thyroid metabolism that were carried out with mass spectrometry, with special focus on the use of this technique for the quantification of molecules involved in thyroid diseases

Presence in the pre-surgical fine-needle aspiration of potential thyroid biomarkers previously identified in the post-surgical one.

Fine-needle aspiration biopsy (FNA) is usually applied to distinguish benign from malignant thyroid nodules. However, cytological analysis cannot always allow a proper diagnosis. We believe that the improvement of the diagnostic capability of pre-surgical FNA could avoid unnecessary thyroidectomy. In a previous study, we performed a proteome analysis to examine FNA collected after thyroidectomy. With the present study, we examined the applicability of these results on pre-surgical FNA. We collected pre-surgical FNA from 411 consecutive patients, and to obtain a correct comparison with our previous results, we processed only benign (n=114), papillary classical variant (cPTC) (n=34) and papillary tall cell variant (TcPTC) (n=14) FNA. We evaluated levels of five proteins previously found up-regulated in thyroid cancer with respect to benign nodules. ELISA and western blot (WB) analysis were used to assay levels of L-lactate dehydrogenase B chain (LDHB), Ferritin heavy chain, Ferritin light chain, Annexin A1 (ANXA1), and Moesin in FNA. ELISA assays and WB analysis confirmed the increase of LDHB, Moesin, and ANXA1 in pre-surgical FNA of thyroid papillary cancer. Sensitivity and specificity of ANXA1 were respectively 87 and 94\% for cPTC, 85 and 100\% for TcPTC. In conclusion, a proteomic analysis of FNA from patients with thyroid nodules may help to distinguish benign versus malignant thyroid nodules. Moreover, ANXA1 appears to be an ideal candidate given the high sensitivity and specificity obtained from ROC curve analysis

Identification and Functional Studies of Two New Dual-Oxidase 2 (DUOX2) Mutations in a Child with Congenital Hypothyroidism and a Eutopic Normal-Size Thyroid Gland

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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

Autoantibodies from patients with autoimmune thyroid disease do not interfere with the activity of the human iodide symporter gene stably transfected in CHO cells

OBJECTIVE: The human sodium iodide symporter (hNIS) is a candidate autoantigen in autoimmune thyroid diseases. To investigate the possible existence of autoantibodies able to interfere with the biological activity of hNIS, an assay was developed using a cell line stably expressing hNIS. METHODS: hNIS complementary cDNA cloned in pcDNA3 and a neomycin resistance gene vector were co-transfected into CHO cells. After selection with geneticin, a cell line termed PA4, showing the highest level of Na(125)I uptake, was characterized. The time course of iodide uptake was evaluated by incubating PA(4) cells with 10 micromol/l NaI and 0.1 microCi Na(125)I for a period up to 90 min. The accumulation of iodide increased linearly between 2 and 10 min, reaching a plateau at 45 min. The curve of iodide efflux mirrored that of iodide influx. Both perchlorate and thiocyanate inhibited iodide uptake in PA(4) cells in a dose-dependent manner starting from concentrations as low as 0.01 and 0.1 micromol/l respectively and complete inhibition was obtained at concentrations of 100 micromol/l perchlorate and 1000 micromol/l thiocyanate. The sensitivity of the inhibition assay was further improved using both inhibitors after 5 min incubation and in the absence of cold NaI. RESULTS: Included in the study were 42 patients with Graves' disease (25 had active hyperthyroidism, ten were euthyroid and seven had hypothyroidism); 34 patients with Hashimoto's thyroiditis (one was euthyroid, four had subclinical hypothyroidism and 29 were overtly hypothyroid); and 19 with atrophic thyroiditis (all hypothyroid). Four out of eight whole sera from patients with Hashimoto's thyroiditis, and 8 out of 25 whole sera from patients with Graves' disease caused an inhibition of iodide uptake in PA(4) cells greater than 20% but also in 4 out of 15 sera from normal subjects. This inhibition activity exerted by sera from patients and controls was lost after dialyzing against buffer. Accordingly, IgGs purified from sera of all patients with Graves' disease and with Hashimoto's thyroiditis or atrophic thyroiditis were devoid of any effect on iodide uptake. CONCLUSIONS: In conclusion, we believe that autoantibodies able to block the function of hNIS are very rare

Constitutive Activation of the Thyroid-Stimulating Hormone Receptor (TSHR) by Mutating Ile691 in the Cytoplasmic Tail Segment

/inositol phosphate (IP) pathways, which stimulate thyroid hormone production and thyroid proliferation./IP signaling pathway./IP cascade

TSH elevations as the first laboratory evidence for pseudohypoparathyroidism type Ib (PHP-Ib).

Hypocalcemia and hyperphosphatemia because of resistance toward parathyroid hormone (PTH) in the proximal renal tubules are the most prominent abnormalities in patients affected by pseudohypoparathyroidism type Ib (PHP-Ib). In this rare disorder, which is caused by GNAS methylation changes, resistance can occur toward other hormones, such as thyroid-stimulating hormone (TSH), that mediate their actions through G protein-coupled receptors. However, these additional laboratory abnormalities are usually not recognized until PTH-resistant hypocalcemia becomes clinically apparent. We now describe four pediatric patients, first diagnosed with subclinical or overt hypothyroidism between the ages of 0.2 and 15 years, who developed overt PTH-resistance 3 to 20 years later. Although anti-thyroperoxidase (anti-TPO) antibodies provided a plausible explanation for hypothyroidism in one of these patients, this and two other patients revealed broad epigenetic GNAS abnormalities, which included loss of methylation (LOM) at exons AS, XL, and A/B, and gain of methylation at exon NESP55; ie, findings consistent with PHP-Ib. LOM at GNAS exon A/B alone led in the fourth patient to the identification of a maternally inherited 3-kb STX16 deletion, a well-established cause of autosomal dominant PHP-Ib. Although GNAS methylation changes were not detected in additional pediatric and adult patients with subclinical hypothyroidism (23 pediatric and 39 adult cases), hypothyroidism can obviously be the initial finding in PHP-Ib patients. One should therefore consider measuring PTH, along with calcium and phosphate, in patients with unexplained hypothyroidism for extended periods of time to avoid hypocalcemia and associated clinical complications

Assessment of sense of taste in Italian Centenarians

The Italian multicentric study on centenarians (IMSC) was aimed at assessing the level of preservation of the sense of taste, and at estimating to what extent the recognition of various gustative stimuli can give satisfaction and information regarding the surrounding environment for the centenarians. Taste sensitivity has been qualitatively established in a group of 126 Italian centenarians (mean age 101.9 \ub1 1.4 years) and compared to that of a group of 100 elderly subjects (mean age 70.5 \ub1 5.0 years). All the individuals included in the study groups had a mini mental state examination (MMSE) score larger than 19. The results revealed that taste sensitivity is significantly reduced in the centenarians; as a matter of fact, the elderly control subjects are able to perceive taste stimuli in 84.25 \ub1 3.40% of the testing, while the centenarians were successful only in 74.23 \ub1 6.19% of the experiments (P < 0.001). Furthermore, elderly subjects can correctly recognize taste stimuli in 70% of the testings, while correct recognition amounted only to 49.25% in cases of centenarians assessed. In spite of these differences between the elderly and centenarians, the latter are still able to perceive and recognize taste stimuli adequately, and as a consequence, are able to obtain information on tastes and receive a sufficient sensory stimulation through the tasting pathway

Laboratory parameters of italian centenarians

A consortium of 20 university departments of geriatrics and gerontology conducted the Italian Multicentric Study on Centenarians (IMSC), in order to assess the socio-economic, clinical and biological conditions of the Italian centenarians. The investigation involved 382 subjects randomly selected from a total of 1162 centenarians (234 men and 928 women), recorded by a census carried out until 31 December, 1993. Their case history, clinical and socio-economic data were recorded on a computerized clinical case sheet. Blood samples for the purpose of the present investigations were drawn from 257 of them. A great proportion (79%) of these latter subjects displayed satisfactory general conditions in their laboratory parameters, 18.3% of them had fairly good clinical conditions even with slightly modified laboratory parameters. Only a low percentage (2.8%) had poor general conditions with a marked anemia, hyperazotemia and uric acid levels. Long duration diabetes was practically absent, and only 5.5% of our centenarians displayed hyperglycemia with a mean duration time of 9.3 years. The prevalence of subjects with hypercholesterolemia was 31.1%. Only 4.3% of centenarians was affected by mixed form of dyslipidemia (hypertriglyceridemia associated with hypercholesterolemia), confirming that elevated blood lipid contents jeopardize really long survival