MECHANISMS IN ENDOCRINOLOGY: Impact of isolated TSH levels in and out of normal range on different tissues.

Routine treatment of thyroid cancer (TC) includes long-term suppression of TSH. The necessity of this treatment in low- and intermediate-risk patients as well as the extent of TSH suppression is currently under discussion. A literature search was performed to illustrate the role of TSH in extrathyroidal cells and to identify potential reasons for different effects of exogenously suppressed and endogenously low TSH levels. Although adverse effects of subnormal and supranormal TSH blood levels on heart and brain have not been consistently found, studies show a clear negative effect of suppressed TSH levels on bone mineral density. Experimental data also support an important role of TSH in the immune system. The ability of levothyroxine (l-T4) to regulate TSH levels and triiodothyronine levels in a physiological manner is limited. Reduction of circadian changes in TSH levels, decrease of thyroid hormone-binding proteins, prevention of potential compensatory increases of TSH levels (e.g., in old age), and unresponsiveness of TSH-producing cells to TRH on l-T4 treatment might cause adverse effects of suppressed TSH levels. In view of the adverse effects of aggressive TSH suppression, achieving the suggested levels of TSH between 0.9 and 1 mU/l in the treatment of low-to-intermediate risk TC patients appears justified.

Anti-ruthenium antibodies mimic macro-TSH in electrochemiluminescent immunoassay.

BACKGROUND: Macro-hormones are circulating conjugates of hormones with immunoglobulins, which often artefactually elevate biochemical test results. Particularly when causing only moderate elevation no suspicion will be raised. By far the most frequently encountered macro-hormone is macro-prolactin. Here we report a female patient with rheumatoid arthritis who had persistently and grossly elevated thyroid stimulating hormone (TSH) but normal free thyroxine in electrochemiluminescent assays. Although clinically euthyroid, she was put on thyroxine therapy which caused hyperthyroid symptoms. METHODS: An analytic interference by macro-TSH was assumed by dilution experiments, polyethylene-glycol-precipitation, the addition of a heterophilic antibody blocking reagent and size exclusion chromatography. RESULTS: Further workup, however, revealed the presence of anti-ruthenium antibodies. CONCLUSIONS: To our knowledge this is the first report of anti-ruthenium antibodies selectively interfering with a TSH assay and causing erratic gross elevation of TSH mimicking macro-TSH.

Practical reversed-phase high-performance liquid chromatography method for laboratory-scale purification of recombinant human thyrotropin.

A small, semi-preparative C(4) RP-HPLC column was used to set up the conclusive laboratory-scale purification of Chinese hamster ovary-derived human thyrotropin (hTSH), after a preliminary concentration-purification of an extremely dilute and poorly ( approximately 0.6 microg hTSH/mL; mass fraction=0.35%) conditioned medium on a cation exchanger. Several fractions of this eluate were repeatedly injected on the semi-preparative column, obtaining, in a single run (<1h chromatographic time), a concentrated pool ( approximately 1.2 mg/mL) of highly purified hTSH that could be further concentrated to >3 mg/mL and then efficiently lyophilized. The overall recovery in the rapid RP-HPLC purification step, including concentration and lyophilization, was of the order of 80%. The final product, when tested via a precise, single-dose in vivo bioassay, confirmed that it did not suffer any loss of bioactivity. This same methodology can be easily adapted to the small-scale purification of other recombinant products, even when obtained from genetically modified organisms at extremely low concentrations and mass fractions.

Expression and purification of feline thyrotropin (fTSH): immunological detection and bioactivity of heterodimeric and yoked glycoproteins.

The goal of this study was to express and purify recombinant feline TSH as a possible immunoassay standard or pharmaceutical agent. Previously cloned feline common glycoprotein alpha (CGA) and beta subunits were ligated into the mammalian expression vector pEAK10. The feline CGA-FLAG and beta subunits were cloned separately into the pEAK10 expression vector, and transiently co-transfected into PEAK cells. Similarly, previously cloned and sequenced yoked (single chain) fTSH (yfTSH) and the CGA-FLAG sequences were ligated into the same vector, and stable cell lines selected by puromycin resistance. Expression levels of at least 1 microg/ml were achieved for both heterodimeric and yoked fTSH forms. The glycoproteins were purified in one step using anti-FLAG immunoaffinity column chromatography to high purity. The molecular weights of feline CGA-FLAG subunit, beta subunit and yfTSH were 20.4, 17, and 45 kDa, respectively. Both heterodimeric and yoked glycoproteins were recognized with approximately 40% detection by both a commercial canine TSH immunoassay and an in-house canine TSH ELISA. The yoked glycoprotein exhibited parallelism with the heterodimeric form in the in-house ELISA, supporting their possible use as immunoassay standards. In bioactivity assays, the heterodimeric and yoked forms of fTSH were 12.5 and 3.4% as potent as pituitary source bovine TSH at displacing (125)I-bTSH and 45 and 24% as potent in stimulating adenylate cyclase activity in human TSH receptor-expressing JP09 cells. However, in addition to reduced receptor binding affinity, the recombinant glycohormones produced a reduced maximal effect at maximal concentration (E(max)) suggesting the possibility of the recombinant glycohormone constructs acting as partial agonists at the human TSH receptor.

Two-step chromatographic purification of recombinant human thyrotrophin and its immunological, biological, physico-chemical and mass spectral characterization.

A purification strategy for rapidly obtaining recombinant human thyrotropin (rhTSH) was designed based on size exclusion and reversed-phase high-performance liquid chromatographic (HPLC) analysis, carried out on hTSH-secreting CHO cell conditioned medium. These analyses permitted the identification of the main contaminants to be eliminated. Considering that hTSH is highly hydrophobic and elutes only with the addition of organic solvents, hydrophobic interaction chromatography was adopted as the first purification step; this resulted in the elimination of, among others, the major contaminant. A second purification step, based on size exclusion chromatography, was then utilized, being effective in the elimination of other previously identified contaminating proteins. Useful purity, as high as 99% at the chemical reagent level, and recoveries (37%) were obtained by adopting this two step strategy, which also provided adequate material for physico-chemical, immunological and biological characterization. This included matrix-assisted laser desorption ionization time-of-flight mass spectral analysis (MALDI-TOF-MS), Western blotting analysis, in vivo biological assay, size-exclusion HPLC (HPSEC) and reversed-phase HPLC (RP-HPLC) analysis, which confirmed the integrity and bioactivity of our rhTSH in comparison with the only two reference preparations available at the milligram level of native (hTSH-NIDDK) and recombinant (Thyrogen) hTSH. Thyrogen and rhTSH-IPEN, when compared to pit-hTSH-NIDDK, presented more than twice as much biological activity and about 7% increased molecular mass by MALDI-TOF-MS analysis, an accurate heterodimer mass determination providing the Mr values of 29,611, 29,839 and 27,829, respectively. The increased molecular mass of the two recombinant preparations was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPSEC analysis. Comparing the two recombinant preparations, minor though interesting physico-chemical and biological differences were also observed.

Radioiodinated recombinant human TSH: a novel radiopharmaceutical for thyroid cancer metastases detection.

UNLABELLED: The follow-up of differentiated thyroid cancer (DTC) is currently performed by serum Tg levels determination and whole body scan (WBS) with 131I. In this regard, the latter represents the main tool to localize metastatic tissue, but is characterized by the induction of severe hypothyroidism. Moreover, WBS displays poor sensitivity in poorly differentiated tumors due to a loss of iodine uptake capacity. AIM: In this study we describe an alternative tracer, radiolabeled rhTSH, for the diagnosis of non-iodine uptaking DTC metastases. METHODS: rhTSH was iodinated with 125I or 123I using an enzymatic method with lactoperoxidase/glucose oxidase. In vitro stability of labeled compounds was assessed in saline and serum and in vivo studies were performed in tumor-bearing nude mice. Three mice were inoculated with ARO cells (TSH receptor negative) and three with PTC-1 cells (TSH receptor positive). After 25 days, mice were injected with 10 microg of 123I-rhTSH (100 microCi) and static images were acquired at 30 minutes, 1, 2, and 3 hours. Animals were then sacrificed and dissected for organ counting. RESULTS: RhTSH was radioiodinated to high specific activity: 132.2 mCi/mg for 123I-rhTSH, 94.3 for 125I-rhTSH. In vitro stability tests revealed no significant release of radioiodine. A clear tumor uptake was detectable after 2 hours in all animals implanted with PTC-1. CONCLUSION: Results obtained so far suggest that radiolabeled rhTSH might be a promising radiopharmaceutical for diagnosis and follow-up of DTC.

Lectin analyses of glycoprotein hormones in patients with congenital disorders of glycosylation.

OBJECTIVE: The congenital disorders of glycosylation (CDGs) are progressive multisystemic disorders characterized by a heterogeneous deficiency of the carbohydrate moieties in various structural and circulating glycoproteins, representing a natural model for glycoprotein hormone studies. Here, we studied the carbohydrate moiety of circulating glycoprotein hormones in four patients with a clinical suspicion of CDGs. METHODS: The diagnosis of CDG-I was confirmed in two out of the four cases by transferrin isoelectrofocusing (IEF) and/or carbohydrate-deficient transferrin (CDT) test. The carbohydrate moiety of serum endocrine-related glycoproteins was investigated by means of Ricin (immunopurified thyrotropin (TSH)) and Concanavalin A (Con-A) (TSH, follicle-stimulating hormone, alpha-subunit and thyroglobulin) lectin affinity chromatography measurement. RESULTS: CDT concentrations were very high in the two patients with CDG-I and moderately enhanced in the remaining two. In the two CDG-I patients, Ricin analysis of immunopurified TSH showed a severe impairment of lectin binding, both before and after neuroaminidase treatment, indicating a nearly complete lack of terminal sialic acid and galactose residues. In these two cases, Con-A analysis showed a significant prevalence of firmly bound isoforms with poorly processed carbohydrate chains. In the remaining two cases with unknown CDG classification, TSH binding pattern to Ricin was modestly affected and Con-A analysis showed the prevalence of weakly bound glycoprotein isoforms. CONCLUSIONS: The results of Ricin analyses in all four patients were consistent with the CDT test and/or serum transferrin IEF. The severe alteration of TSH binding pattern to Ricin seems to be characteristic of CDG-I. Nevertheless, TSH biological properties are not severely altered, as normal thyroid function was found in both cases.

A thyrotropin-like molecule from the pituitary of an Indian freshwater murrel: comparison of its biological activity with other thyrotropins.

Murrel pituitary thyrotropin-like molecule (mTSH) was purified to homogeneity with the help of a convenient and sensitive in vitro assay system where addition of this material to the thyroid follicle incubation stimulated thyroxine (T(4)) secretion into the medium. Pituitary extract of a freshwater murrel, Channa punctatus, was solvent extracted to obtain glycoprotein enriched fraction. This was subjected to Sephadex G-100 gel filtration and eluate of void volume (peak I) showed strong TSH activity (as reflected from T(4) secretion) which was further purified by using concanavalin A-Sepharose, FPLC Mono Q and immunoaffinity chromatography. Purified mTSH gave a single band in PAGE, and SDS PAGE revealed two dissimilar subunits, alpha and beta. Addition of increasing concentrations of mTSH, Indian carp TSH (cTSH) and bovine TSH (bTSH) to in vitro murrel thyroid follicle incubations caused a linear increase in thyroxine (T(4)) release into the medium, effect was highest with mTSH and lowest with bTSH. However, in in vivo experiments, injections of increasing doses of mTSH to murrel elevated plasma T(4) level in a linear manner while bTSH gave a biphasic response. Addition of mTSH and bTSH to rat or goat thyroid epithelial cell incubations equally stimulated T(4) release into the medium, while cTSH had significantly less effect. Binding affinity (K(a)) and receptor occupancy (B(max)) of mTSH to murrel thyroid follicular membrane preparation was considerably higher in comparison to cTSH or bTSH whereas both mTSH and bTSH had nearly similar K(a) and B(max) with rat thyroid epithelial cell membrane preparation. Findings indicate that mTSH is a more potent TSH as compared to carp and bovine TSH in murrel and has equipotent biological activity as bTSH on rat and goat thyroid.

Characterization of epitope regions of thyrotropin beta-subunit recognized by the monoclonal antibodies mAb279 and mAb299: a chimeric peptide approach.

This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin beta-subunit, bTSHbeta. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSHbeta developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised beta-turn regions of loop L1 [bTSHbeta(14-20)] and loop L3 [bTSHbeta(65-72)] held in close proximity by a bis-beta-alanine linker and the disulfide bond bTSHbeta[Cys16-Cys67]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSHbeta [125I-bTSHbeta] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSHbeta that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of 125I-bTSHbeta to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSHbeta-epitope recognized by mAb299 involves contributions from amino residues from the beta-turn regions of the L1 and L3 loops of TSHbeta, and that these loop regions flank part of the receptor binding site of the bTSH beta-subunit.

Evaluation of a sensitive, in vitro bioassay for chicken thyroid stimulating hormone using FRTL-5 cells, a rat thyroid cell line.

An in vitro bioassay for mammalian thyroid stimulating hormone (TSH) based on TSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production in FRTL-5 cells, a rat thyroid cell line, was used to measure chicken TSH. The addition of chicken pituitary homogenate equivalent to > or = 25% of a chicken pituitary gland to cultured FRTL-5 cells increased cAMP within these cells in a dose-dependent manner. The glycoprotein fraction derived from the pituitary homogenate was further fractionated by isoelectric focusing within a pH range of 5 to 11. Analysis of the focused fractions by the bioassay detected three major components with isoelectric points of 9.30, 7.12, and 3.82, in addition to several minor ones distributed over a wide range of pH, from alkaline to acidic. The isoelectric focusing profile obtained by the bioassay was clearly different from those obtained by radioimmunoassay for chicken LH and radioreceptor assay for chicken FSH, indicating that fractions contained chicken TSH. The homogenate of the cephalic portion of the chicken anterior pituitary gland was 4.46 times more active than that of the caudal portion in the bioassay, which is consistent with previous findings on localization of TSH in the chicken pituitary. We conclude that the bioassay using FRTL-5 rat thyroid cells is a sensitive, specific, and time-saving method of measuring chicken TSH.

Hormona estimulante de la tiroides

La hormona estimulante de la tiroides- TSH por throid stimulating hormone-, liberada por la hipófisis anterior, es principal regulador de la función tiroidea. La secreción de la TSH es controlada por el hipotálmo mediante la hormona liberadora de tirotropina - TRH por thyrotropin releasing hormone-. A su vez, este sistemea regula la liberación de las hormonas tiroides: Tiroxina- T4- y tirotropina- T3-. En la producción de la TSH existe un mecanismo de retroalimentación negativo, que es sensible a las concentraciones de las hormonas tiroideas circulantes, T3 y T4, controladas por el hipotálamo. Colectivamente este sistema se denomina eje hipotálamo-hipofisiario-tiroideo. Cualquier alteraciónen la función del eje influye sobre los niveles sanguíneos de la T4 y T3.

Isolation and partial characterization of LH, FSH and TSH from canine pituitary gland.

A new preparative procedure without using ion-exchanger is described for the efficient purification of canine LH (cLH), FSH (cFSH) and TSH (cTSH) from the pituitary gland. The hormones were extracted from the pituitary homogenate with an ammonium sulfate solution, and were separated by Concanavalin (Con) A affinity-, hydrophobic interaction-, then immobilized metal ion affinity chromatography. In the immobilized metal ion affinity chromatography, we used copper (Cu2+) as chelated metal ion with ammonium ion gradient and pH gradient in phosphate buffer to attain separation of the hormones. High purity of cLH, cFSH and cTSH was indicated as single bands in SDS-PAGE, with apparent molecular masses of 34, 36 and 37 kDA, respectively. The purified hormones showed two bands corresponding to alpha (20 kDa) and beta subunits (cLH beta: 16 kDa, cFSH beta: 22 kDa, cTSH beta: 16 kDa) under reducing condition in SDS-PAGE. The purified hormones were prepared in good recovery (LH: 53%, FSH: 34%, TSH: 36%) with high biological activity or binding activity to the receptor. Cross-contamination of the purified hormone was less than 0.5%. Examination of the hormone fraction with isoelectric focusing showed that major peaks of isoelectric isoforms were maintained throughout the purification steps of cLH and cFSH, while a few peaks were lost in Con A affinity chromatography in cTSH purification. It was concluded that the present method could prepare highly purified cLH, cFSH and cTSH which retained isoforms of the hormones and biological activity or binding affinity to the receptor.

Salmon thyroid-stimulating hormone: isolation, characterization, and development of a radioimmunoassay.

Coho salmon (Oncorhynchus kisutch) thyroid-stimulating hormone (TSH) was isolated by ethanol extraction of pituitary glands from mature coho salmon. Extraction was followed by gel-filtration chromatography on Sephadex G-100 superfine, anion-exchange chromatography on a Whatman DE-52 column, and finally by reverse-phase high-performance liquid chromatography. Fractions were monitored for TSH content by a homologous in vivo bioassay and by immunoblots using anti-human TSH beta-subunit antisera. In vivo treatment of coho salmon parr with coho salmon TSH caused a dose-dependent increase in plasma thyroxine level similar to that induced by bovine TSH. The N-terminal sequence (25 residues) of the salmon TSH beta subunit has 56% sequence identity to that of human TSH beta subunit and is identical to the deduced amino acid sequence of trout TSH beta subunit. The N-terminal sequence (25 residues) of the salmon TSH alpha subunit is identical to gonadotropin alpha-II subunit. Molecular sizes of the alpha and beta subunits are 18,000 and 24,000 daltons, respectively, as estimated by SDS-PAGE. Antiserum generated against salmon TSH, which was preadsorbed with alpha subunit using an alpha-subunit affinity column, detected only salmon TSH beta subunit by immunoblot and specifically stained thyrotropin-producing cells of the pituitary gland. A homologous radioimmunoassay (RIA) was developed using purified salmon TSH standard, iodinated TSH beta subunit, and antiserum generated against salmon TSH. Cross-reactivities of GTH I, GTH II, GTH I beta and GTH II beta subunits, alpha subunit, growth hormone, prolactin, and somatolactin were less than 1%. Displacement curves for serial dilutions of plasma and pituitary extracts of various salmonid species, as well as coho salmon pituitary cell culture medium, were parallel to the coho salmon TSH standards. In contrast, plasma of hypophysectomized juvenile coho salmon and pituitary extracts of Pacific tomcod (Microgadus proximus) did not displace bound radiolabeled salmon TSH. Finally, in vivo injection of juvenile coho salmon with triiodothyronine decreased plasma TSH levels, whereas the goitrogen, methimazole, increased plasma TSH levels. Injection of gonadotropin-releasing hormone agonist did not alter plasma TSH. These data suggest that the RIA is specific for TSH and confirm a negative-feedback relationship between the thyroid hormones and TSH.

High-affinity and specific recognition of human thyroid stimulating hormone (hTSH) by in vitro-selected 2'-amino-modified RNA.

RNA sequences containing 2'-amino pyrimidines that bind with high-affinity to human thyroid stimulating hormone (hTSH) were isolated from a random sequence library by an in vitro selection-amplification procedure. A representative RNA ligand (T-15) has an equilibrium dissociation constant (Kd) of 2.5 nM for its interaction with hTSH and can discriminate between other members of the glycohormone family; no detectable binding was observed at low micromolar concentrations of hCG (human chorionic gonadotropin), while measured Kd values for the interactions with hLH (human leutinizing hormone) and hFSH (human follicle stimulating hormone) were > 1 microM and approximately 0.2 microM, respectively. The detection of hTSH in a dot blot assay with radiolabeled T-15 RNA was demonstrated.

Localization of macrophage migration inhibitory factor (MIF) to secretory granules within the corticotrophic and thyrotrophic cells of the pituitary gland.

BACKGROUND: Macrophage migration inhibitory factor (MIF) was one of the first lymphokine activities to be discovered and was described almost 30 years ago to be a soluble factor(s) produced by activated T lymphocytes. In more recent studies, MIF has been "rediscovered" to be an abundant, pre-formed constituent of the anterior pituitary gland and the macrophage, and to be a critical component in the host response to septic shock. Pituitary-derived MIF enters the circulation after infectious or stressful stimuli and appears to act to counterregulate glucocorticoid suppression of cytokine production. MATERIALS AND METHODS: Immunoelectron microscopy utilizing a combination of anti-MIF and anti-pituitary hormone-specific antibodies was used to study the ultrastructural localization of MIF within the anterior pituitary gland. Pituitaries were obtained from resting, unstimulated mice and from mice 16 hr after endotoxin administration. The release of MIF also was investigated in vitro by examining the effect of corticotropin-releasing hormone (CRH_ on the AtT-20, corticotrophic cell line. RESULTS: MIF localizes to granules present exclusively in ACTH and TSH secreting cells. Within each cell type, a subset of granules was found to contain both MIF and ACTH, or MIF and TSH. The pituitary content of MIF-containing granules decreased significantly after experimentally induced endotoxemia. In seven pituitaries examined 16 hr after LPS injection, the number of MIF-positive granules diminished by 38% in corticotrophic cells and by 48% in thyrotrophic cells when compared with controls (p < 0.05). CRH was observed to be a potent MIF secretagogue in vitro, inducing the release of MIF from corticotrophic cells at concentrations lower than that required for ACTH release. CONCLUSION: These data provide ultrastructural information that identify MIF to be a novel anterior pituitary hormone, support earlier studies showing a time-dependent release of pituitary MIF during endotoxemia, and suggest an important, systemic role for MIF in the stress response to infection and other stimuli.

[Clinical aspects, diagnosis and drug therapy of hyperthyroidism]. / Klinik, Diagnostik und medikamentöse Therapie der Hyperthyreose.

Graves' disease and toxic uni- or multinodular goiter are the most frequent causes of hyperthyroidism. Graves' disease is caused by thyroid stimulating immunoglobulins which are directed against the TSH receptor of thyroid follicular cells. Graves' disease affects more females than males and is associated with diffuse goiter and a rapid appearance of symptoms and signs of hyperthyroidism. Patients with Graves' disease are on average younger than patients with toxic nodular goiter. The diagnosis of Graves' disease is usually easy, particularly if signs of endocrine opthalmopathy are present. Toxic nodular goiter is seen more often in older patients with pre-existing goiters. Symptoms and signs of hyperthyroidism often appear only slowly. Hyperthyroidism in these older patients can be oligosymptomatic. Older patients should therefore be investigated for the presence of hyperthyroidism, even if they present only a few symptoms or signs which could suggest this diagnosis. The development of ultrasensitive TSH assays has simplified the diagnosis of hyperthyroidism and made the TRH-test, often used in the past, almost superfluous. At the present time, it is practically always possible to differentiate between Graves' disease and toxic nodular goiter as the cause of hyperthyroidism on the basis of clinical and laboratory findings alone, and in many cases thyroid scintiscans are therefore no longer necessary. A patient with newly diagnosed Graves' disease is treated with antithyroid drugs (carbimazole or PTU) for one year. If hyperthyroidism persists after this one year of antithyroid drug treatment, or if it recurs, another year of therapy with carbimazole or PTU is indicated.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the leakage of a monoclonal antibody from various immunoaffinity chromatography matrices.

Antibody leakage from immunoaffinity chromatography (IAC) matrices could reduce the working life of the IAC matrix and/or contaminate parenteral products, purified by IAC. There is therefore a need to measure the leakage of antibody from IAC matrices and to reduce such leakage. Using sensitive ELISAs it was found that the type of activated matrix, the buffer, the presence of proteases in the feedstock and the storage of IAC matrices between runs could all effect antibody leakage.

Serum thyrotropin (TSH) heterogeneity in euthyroid subjects and patients with subclinical hypothyroidism: the core fucose content of TSH-releasing hormone-released TSH is altered, but not the net charge of TSH.

The aims of the present study were to determine the influence of brief subclinical hypothyroidism on the isoforms of serum thyrotropin (TSH) and to examine the net charge of TSH in different metabolic states. Sera were obtained from euthyroid subjects (n = 7) and from patients with subclinical hypothyroidism (n = 8) before and 30 min after the intravenous administration of 200 micrograms thyrotropin-releasing hormone (TRH). The TSH from human pituitary extracts (IRP 68/38), basal and TRH-stimulated serum TSH was immunoconcentrated and further analysed by isoelectric focusing (IEF) and lentil lectin affinity chromatography. TSH immunoreactivity was determined in each specimen or fraction with an automated highly sensitive chemiluminometric TSH assay. We found that basal TSH in subclinical hypothyroidism, and TRH-released TSH in euthyroidism and in subclinical hypothyroidism is distributed in a similar neutral to acidic pattern, which significantly differs from the more alkaline to neutral isoform pattern of intrapituitary TSH (P < 0.05). IEF analysis of pituitary standard TSH revealed 3 major peaks (pI values 7.5; 6.6; 5.8) whereas in most euthyroid or subclinically hypothyroid subjects 5 peaks were found. Lentil lectin affinity chromatography revealed that TRH-released TSH in euthyroid subjects has more core fucose residues than TSH from patients with subclinical hypothyroidism (64.6 +/- 6.7 vs 12.5 +/- 2.7%, P < 0.0001). Thus pituitary standard TSH seems to be less mature material than circulating TSH. Perhaps no alteration in the IEF pattern of TSH was detected during early hypothyroidism because sialylation of TSH was increasing as sulfatation was decreasing. Nevertheless, a change in the core fucose content of TSH was detectable by lentil analysis.

Pesquisa neonatal de hipotiroidismo congénito (HC) : experiencia en sangre de cordón / Neonatal search of congenital hypothyroidism (CH) : experience in blood cord

Introducción: La imposibilidad de llevar a cabo la pesquisa de hipotiroidismo congénito en el momento del alta de la maternidad llevo a implementar la pesquisa en sangre de cordón a fin de prevenir la discapacidad mental que esta enfermedad produce de no diagnosticarse y tratarse adecuadamente en forma precoz. Objetivo: El objetivo de este trabajo es relatar la experiencia en la pesquisa neonatal de hipotiroidismo congénito (HC) realizada en sangre de cordón implementada en la Maternidad Sardá desde el 1/10/1991 hasta el 31/3/1995. Población y métodos: Se estudiaron 22.384 recién nacidos (82.6 por ciento de término y 17.4 por ciento prematuros). TSH se determinó por métodos inmunoradiométrico (IRMA) e inmunofluorométrico (IFMA) en sangre seca en papel de filtro obtenida de cordón umbilical. Resultados: Se detectaron 6 HC permanentes y 2 transitorios. Incidencia 1:3.790 y 1:11.192 respectivamente. La cobertura, de 80 por ciento en un comienzo alcanzó el 99.5 por ciento en 1995. La edad de confirmación diagnóstica y tratamiento fue de 19ñ5 días (XñDS). El tiempo de ubicación varió entre 1 y 14 días y fue el factor negativo en la implementación del programa. Todos los niños fueron seguidos y maduran normalmente. Conclusión: La pesquisa de HC en sangre de cordón, en el contexto de un programa de pesquisa, constituye una alternativa válida de diagnóstico para esta enfermedad inaparente en el momento del nacimiento.

Pesquisa neonatal de hipotiroidismo congénito (HC) : experiencia en sangre de cordón / Neonatal search of congenital hypothyroidism (CH) : experience in blood cord

Introducción: La imposibilidad de llevar a cabo la pesquisa de hipotiroidismo congénito en el momento del alta de la maternidad llevo a implementar la pesquisa en sangre de cordón a fin de prevenir la discapacidad mental que esta enfermedad produce de no diagnosticarse y tratarse adecuadamente en forma precoz. Objetivo: El objetivo de este trabajo es relatar la experiencia en la pesquisa neonatal de hipotiroidismo congénito (HC) realizada en sangre de cordón implementada en la Maternidad Sardá desde el 1/10/1991 hasta el 31/3/1995. Población y métodos: Se estudiaron 22.384 recién nacidos (82.6 por ciento de término y 17.4 por ciento prematuros). TSH se determinó por métodos inmunoradiométrico (IRMA) e inmunofluorométrico (IFMA) en sangre seca en papel de filtro obtenida de cordón umbilical. Resultados: Se detectaron 6 HC permanentes y 2 transitorios. Incidencia 1:3.790 y 1:11.192 respectivamente. La cobertura, de 80 por ciento en un comienzo alcanzó el 99.5 por ciento en 1995. La edad de confirmación diagnóstica y tratamiento fue de 19ñ5 días (XñDS). El tiempo de ubicación varió entre 1 y 14 días y fue el factor negativo en la implementación del programa. Todos los niños fueron seguidos y maduran normalmente. Conclusión: La pesquisa de HC en sangre de cordón, en el contexto de un programa de pesquisa, constituye una alternativa válida de diagnóstico para esta enfermedad inaparente en el momento del nacimiento. (AU)