Recombinant human thyroid-stimulating hormone (Thyrogen) in thyroid cancer follow up: experience at a single institution.

BACKGROUND: Recombinant human thyroid-stimulating hormone (Thyrogen; Genzyme Corporation, Cambridge, MA, USA) (rhTSH)-stimulated serum thyroglobulin (Tg) (stim-Tg) and (131)I whole-body scanning (WBS) have been reported to allow follow up of patients with thyroid cancer without the symptoms of thyroxine withdrawal and with equivalent diagnostic information to that obtained after thyroxine withdrawal. The aim of the study was to report results of rhTSH use at the Alfred Hospital, Melbourne, from 1999 to 2006 and in particular to examine the significance of detectable serum Tg after rhTSH in relation to thyroid cancer staging and to compare the sensitivity of rhTSH-stimulated serum Tg to whole-body (131)I scanning (WBS) in the detection of residual and recurrent thyroid cancer. METHODS: The study was a retrospective chart review. RESULTS: In 90 patients, rhTSH was used for 96 diagnostic episodes and 18 doses of rhTSH were used to facilitate treatment with (131)I. In stages I and II cancer (n = 42), of three patients with stim-Tg 1-2 microg/L, none had identifiable disease, and the three patients who had stim-Tg >2 microg/L did not experience recurrent disease during follow up. In contrast, in stages III and IV cancer (n = 43) 2 of 5 with stim-Tg 1-2 microg/L had identifiable disease and 7 of 10 with stim-Tg >2 microg/L had identifiable disease. In Tg-positive, WBS-negative disease, further imaging identified persistent/recurrent disease. CONCLUSION: rhTSH was effective and safe in the management of thyroid cancer follow up for diagnosis of persistent/recurrent cancer and to enable (131)I treatment. In no case did rhTSH-stimulated WBS identify the presence of disease not also identified by raised basal Tg or stim-Tg. Therefore, in low risk cancer WBS may be omitted.

Heterogeneity of amiodarone-induced thyrotoxicosis: evaluation of colour-flow Doppler sonography in predicting therapeutic response.

BACKGROUND: Amiodarone-induced thyrotoxicosis (AIT) presents a therapeutic challenge because of its resistance to standard antithyroid therapy. In iodine-deplete environments, colour-flow Doppler sonography (CFDS) has allowed distinction between two types of AIT: (i) Type I AIT, associated with increased vascularity (CFDS I-III) and response to thionamide antithyroid drug and (ii) type II AIT, with no/little thyroid vascularity (CFDS 0) and prednisolone responsiveness. AIM: To clarify if CFDS patterns correlated with treatment outcomes in a retrospective study of 24 patients with AIT in an iodine-replete environment. METHODS: Medical records of patients who presented to a teaching hospital between January 1998 to December 2000 were reviewed. Results of CFDS, ultrasound measurement of thyroid size and technetium scanning of the thyroid were correlated with treatment responses, especially prednisolone responsiveness. RESULTS: Thirteen of 24 patients showed CFDS 0. Twelve of these 13 were evaluable for prednisolone responsiveness, of whom seven (58%) were prednisolone-responsive. Of 11 patients with CFDS I-III, four (36%) responded to antithyroid medication alone and only one of seven (14%) was prednisolone-responsive. Euthyroidism was achieved twice as rapidly in patients with CFDS 0 than those with CFDS I-III. Because of medical treatment failure, seven patients, from both CFDS groups, required urgent near-total thyroidectomy which was successful and uncomplicated in all cases. CONCLUSIONS: CFDS is useful in the management of AIT because CFDS 0 correlates better with prednisolone response (58%) than CFDS I-III (14%). However, unlike experience in iodine-deficient regions, the results of the present study revealed that treatment responses to thionamide or prednisolone were heterogeneous within uniform CFDS patterns. Thus, prednisolone--responsiveness was not consistently predicted by CFDS 0, but the presence of flow appeared to correlate with non-response to prednisolone.

Free thyroid hormone measurement. A critical appraisal.

The main purpose of free T4 and free T3 assays is to distinguish reliably between thyrotoxicosis, hypothyroidism, and the euthyroid state, an objective that cannot be attained with assays of total T4 and T3 because of hereditary and acquired variations in the concentrations of binding proteins. Effective correction for changes in the serum concentration of TBG can be achieved with numerous types of free hormone estimate, but other changes in binding are not well accommodated. Despite remarkable methodologic ingenuity, no current method reflects the free T4 concentration in undiluted serum under in vivo conditions. Equilibrium dialysis, widely considered the reference method for free T4 measurement, is also subject to error, either preanalytic, owing to generation of NEFA in the sample leading to an overestimate of free T4, or analytic with underestimation of the effect of competitors to increase free T4. Current approaches to free T4 measurement are vulnerable to several method-dependent artifacts: abnormal albumin binding of T4 or of the assay tracer, the inhibition of T4 binding to TBG by medications, and the effects of critical illness, especially in heparin-treated patients, pregnancy, and the abnormalities in sick premature infants. Because of systematic variation between methods (i.e., whether a technique is albumin dependent or prone to incubation or dilution artifacts), it is essential to consider methodologic details in evaluating free T4 estimates in these situations and whenever estimates of free T4 are clinically discordant. False-positive abnormalities are more frequent than false-negative results. When free T4 results are correlated with the serum TSH concentration with attention to the assumptions that define this relationship, the majority of false-positive results can be readily identified. If a free T4 anomaly remains unexplained on repeat sampling, it is appropriate to use an alternative free T4 method that depends on a different assay principle and to correlate the result with an authentic total T4 measurement.

Tooth root resorption associated with a familial bone dysplasia affecting mother and daughter.

The dental findings are presented of a mother and daughter who suffer from an as yet unclassified bone dysplasia that shows features of both hereditary hyperphosphatasia and familial expansile osteolysis. Both patients have experienced progressive root resorption of permanent teeth, deafness, and high alkaline phosphatase levels. The mother has a more advanced bone dysplasia which has led to progressive skeletal deformity and bone pain. The kindred is consistent with an autosomal dominant pattern, and the mutation(s) is thought to be in chromosome 18q21-22 region. Conventional treatment strategies of root resorption offer only a poor prognosis for the dentition. Therapy using alendronate, a bisphosphonate compound and a potent inhibitor of osteoclastic activity, has reduced alkaline phosphatase levels, bone pain, and may offer an effective strategy to prevent tooth root resorption in this group of diseases.

Modulation of T3-induced sex hormone-binding globulin secretion by human hepatoblastoma cells.

We have shown previously that tri-iodothyronine (T3)-induced sex hormone-binding globulin (SHBG) secretion by the human hepatoblastoma cell line, HepG2, can be modulated by retinoids. We have now used this model to study a range of other compounds that are known to influence T3 responsiveness in various cell systems. HepG2 cells were incubated for 4 days in serum-free medium containing T3, together with insulin, dexamethasone, phorbol myristate (PMA), sodium butyrate or estradiol. T3 (10 nmol/l) alone induced a concentration of SHBG secreted by HepG2 cells that was 187 +/- 20% (mean +/- S.D., n = 9) of control. Insulin (100 nmol/l) reduced basal SHBG secretion from 24.7 +/- 5.2 nmol/l to 16.1 +/- 1.7 nmol/l (P < 0.01). This effect was dose responsive, half-maximal at 3.4 +/- 3.0 nmol/l (approximately 600 mU/l) and maximal with 100 nmol/l insulin. Co-incubating 0-10 nmol/l T3 with 100 nmol/l insulin resulted in a downward shift in the dose-response curve without a change in the half-maximal response to T3. Conversely, 0-100 nmol/l insulin reduced SHBG production induced by 10 nmol/l T3. In contrast; while dexamethasone alone was without effect on SHBG secretion, 100 nmol/l dexamethasone induced a shift to the left in half-maximal T3 stimulation from 0.37 nmol/l to 0.10 nmol/l. The effect of PMA on SHBG secretion was reminiscent of the previously observed retinoid effect. PMA 100 nmol/l abolished maximal T3 stimulation. This effect was dose responsive, with a threshold at 1 nmol/l PMA. Sodium butyrate, up to 1 mmol/l was without effect; with greater concentrations, SHBG secretion was reduced. T3 responsiveness was virtually abolished by 3 mmol/l sodium butyrate; higher concentrations were cytotoxic and secretion was reduced to less than 20% of basal. Lack of an effect of estradiol on SHBG secretion by HepG2 cells was confirmed. These studies suggest that T3-induced SHBG secretion by HepG2 cells is independently influenced by insulin, potentiated by dexamethasone, and modulated by PMA. Detailed molecular analysis of this model will increase our understanding of the mechanism of action of T3, specifically in human liver cells.

The commercialisation of public teaching hospitals is a fundamental error.

The new funding system for Australia's public hospitals has not been shown to be effective and is likely to fragment and undermine services that require cooperation rather than competition.

Differential modulation of thyroid hormone responsiveness by retinoids in a human cell line.

Previous studies have suggested that there is an interrelationship between responses mediated by retinoic acid (RA) and those to thyroid hormone (T3). These experiments have used transfected gene constructs, often in receptor-negative cells. To study the relationship between RA- and T3-mediated responses in intact human cells, we incubated HepG2 cells for 4 days in serum-free medium with T3 and/or RA or 9-cis-RA. Measured responses were stimulation of secreted sex hormone-binding globulin (SHBG) or inhibition of secreted T4-binding globulin (TBG). T3 induced a dose-responsive increase in SHBG secretion that was maximal at 10nM (206 +/- 24% of untreated value) and half-maximal at 0.36 +/- 0.16 nM T3. RA and 9-cis-RA, up to 100 nM, induced a slight fall in SHBG secretion to 79 +/- 9% and 88 +/- 9%, respectively. T3 induction of SHBG secretion was significantly attenuated in cells coincubated with T3(0-10nM) and RA. With T3 (10 nM) together with RA (3, 10, or 100 nM), the maximal SHBG responses were reduced to 193 +/- 24%, 151 +/- 5% and 132 +/- 30%, respectively. With T3 and 9-cis-RA (100 nM), maximal stimulation was 169 +/- 20%. Importantly, the effective half-maximal stimulatory concentration of T3 in the presence of either retinoid (3-100 nM) was unchanged at 0.3 nM T3. In addition, the inhibitory effect of 9-cis RA could not be overcome even with 300 nM T3. The threshold for the RA effect was between 0.3-1 nM, with half-maximal inhibition at 30 nM. 9-cis-RA was approximately 10-fold less potent than RA. Preliminary studies suggested that changes in SHBG messenger RNA levels were similar to those in secreted SHBG. No effect was observed with vitamin D or clofibrate, either alone or combined with T3. Conversely, T3 reduced TBG secretion, with maximal suppression to 74 +/- 5% of the control value at a T3 concentration of 10 nM. RA alone reduced TBG secretion to 76% of the control value. RA did not attenuate the effect of T3, and the two agents combined showed no synergism. Neither T3 nor RA, alone or in combination, influenced secreted total protein or albumin. RA did not alter the concentration of nuclear T3-binding sites. These data suggest that retinoids act via a gene-dependent mechanism to modulate maximal, but not half-maximal, responses to T3 in HepG2 cells with the specificity of RA greater than that of 9-cis-RA.

Concentrations of thyroid hormones and iodothyronine binding proteins in serum of the koala (Phascolarctos cinereus).

OBJECTIVE: To examine circulating total and free thyroid hormone (T3 and T4) concentrations, determine serum iodothyronine binding characteristics and estimate thyroid stimulating hormone (TSH) activity in sera of coastal and inland koalas. DESIGN: A prospective study. PROCEDURE: Koala serum T3 and T4 were measured by radioimmunoassay. T4 binding parameters were determined by radioligand binding and electrophoresis. Koala TSH values were determined by bioassay. RESULTS: Mean total T4 concentrations were 3.2 +/- 2.1 nM although values were significantly higher in inland-dwelling females in comparison to coastal-dwelling males. Free T4 was 3.3 +/- 2.1 pM. Total and free T3 were 0.4 +/- 0.2 nM and 1.4 +/- 0.9 pM respectively, although these values were at the lower end of the assay detection limit and should be viewed with reservation. Electrophoresis of [125I]-T4-labelled serum revealed only two proteins of electrophoretic mobility similar to human transthyretin (TTR) and albumin. Scatchard analysis of T4 binding to serum gave a curvilinear plot, which could be resolved into two binding sites with affinities identical to that of TTR and albumin but both of low concentration. The bioactivity of the TSH present in the sera was measured using a cell line (JP09) transfected with the human TSH receptor. The mean level of stimulation found in the sera corresponded to a bovine TSH activity of less than 10 mU/L. CONCLUSION: These results suggest that the serum concentrations of free and total thyroid hormones in koalas are low compared to other marsupials and very low compared to eutherian mammals. The mechanism of maintenance of euthyroidism in this species remains to be determined.

Preferential inhibition of cytoplasmic T3 binding is associated with reduced nuclear binding in cultured cells.

Previous studies from our laboratory have suggested that the nonsteroidal antiinflammatory drug, diclofenac (DCF), is a more potent competitor for T3 binding sites in cytoplasm than for those in the nucleus. In the present study we have examined the competitive potency for DCF and its effect on nuclear binding of T3 in cultured cells. DCF was a weak competitor for T3 binding sites in cytosol and nuclear extracts prepared from HepG2 cells with a potency of 21 and 295 microM, respectively. When expressed relative to T3, DCF was 135-fold more potent in cytosol than in nuclear extract. In intact cells, T3 was bound by nuclei with an affinity, Kd of 0.22 +/- 0.07 nM whereas in nuclear extract the affinity was 0.60 +/- 0.21 nM. DCF was a competitive inhibitor in both preparations but reduced the apparent affinity 4-fold in intact cells but only 2-fold in nuclear extract. In whole-cell experiments, DCF increased the rate of dissociation of T3 from cells prelabeled with hormone for 30 min. When these prelabeled cells were incubated with DCF, 0.1 mM, cell-associated T3 was significantly lower at 30 and 60 min than in cells reincubated without the drug. These data show that cellular transport mechanisms precede nuclear binding by T3 and suggest that there is a critical role for nonnuclear binding proteins in thyroid hormone action.

Guidelines for diagnosis and monitoring of thyroid disease: nonthyroidal illness.

On the basis of low specificity, poor positive predictive value, and cost, there is at present no basis for routine assessment of thyroid function in acutely hospitalized patients, unless clinical features suggest the possibility of thyroid dysfunction, or a patient's background increases the likelihood of thyroid dysfunction. When used in severely ill patients, estimates of both thyroxine (T4) and thyrotropin (TSH) show a high prevalence of abnormal results, but lack specificity and have poor positive predictive value for true thyroid disease. When thyroid function is tested in the critically ill, the positive predictive value for true thyroid disease of both free T4 and TSH measurements could be improved by using wider reference intervals than for unselected populations. The knowledge of nonspecific disease-related abnormalities of triiodothyronine, T4, and TSH is not currently likely to yield useful prognostic information or to alter management for individual patients. Thyroid testing should be readily available for any acutely ill patient with any clinical features that suggest thyroid dysfunction, and for groups at increased risk of thyroid dysfunction. An initial abnormal result for either TSH or free T4 estimate should be followed by combined analysis of free T4 and TSH with the best available methodology. Diagnosis of thyroid dysfunction should be based on the T4-TSH relation rather than either value alone. Persistence of an apparent diagnostic abnormality should be confirmed before therapy is commenced.

Drug effects on triiodothyronine uptake by rat anterior pituitary cells in vitro.

In nonthyroidal illness, numerous drugs such as glucocorticoids, dopamine, fenclofenac, furosemide and diphenylhydantoin may modify the close inverse-feedback relationship between circulating thyroid hormones and TSH. Such effects could involve altered hypothalamic TRH secretion, a direct effect on TSH production by the thyrotroph, alterations in circulating free thyroid hormone concentrations, or changes in thyroid hormone uptake by the thyrotroph. We therefore examined the effect of nonsteroidal antiinflammatory drugs (NSAID), diuretics, the synthetic flavonoid EMD 21388, and diphenylhydantoin, on [125I]T3 cellular uptake in rat pituitary primary cell cultures. Uptake of [125I]T3 (cell-associated counts of washed cells) was measured at 15 min after the addition of 50 pmol/L [125I]T3 in protein-free medium (37 degrees C, pH 7.4). Uptake of [125I]T3 by pituitary cells was 6.0 +/- 1.7% of total counts (mean +/- SD, n = 18). Unlabeled T3 (10 mumols/L) displaced 92% of total uptake. The IC50 of unlabeled T3 for the displacement of [125I]T3 was 1.2 mumols/L. T4 and rT3 were approximately 10% as effective as T3 itself in inhibiting [125I]T3 uptake, while triac did not affect cellular [125I]T3 uptake. Inhibition of [125I]T3 uptake at drug concentrations of 100 mumols/L was seen with the diuretics, furosemide (9%), bumetanide (14%), piretanide (12%) and ethacrynic acid (76%), the NSAID, meclofenamic acid (35%) and fenclofenac (52%), EMD 21388 (49%), and the anticonvulsant, diphenylhydantoin (23%). Aspirin, up to 500 mumols/L, had no effect on [125I]T3 uptake. Our results indicate that ethacrynic acid, meclofenamic acid, fenclofenac, EMD 21388 and diphenylhydantoin affect plasma membrane T3 uptake in the pituitary. This potential influence on TSH release will be contrary to the previously-demonstrated direct inhibitory effect of these drugs on TSH release.

Influence of nonesterified fatty acids and lysolecithins on thyroxine binding to thyroxine-binding globulin and transthyretin.

The hydrolysis of lecithin by phospholipase produces equimolar amounts of nonesterified fatty acids (NEFAs) and lysolecithin. In this study, we have evaluated the effect of lysolecithins and NEFAs on thyroid hormone binding by examining their interactions with thyroxine-binding globulin (TBG)(serum 1:10,000 dilution) and purified transthyretin (TTR). Unsaturated NEFAs (palmitoleic, oleic, linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acid) inhibited [125I]T4 binding to TBG. Their affinities, relative to unlabeled T4, ranged from 0.005 to 0.0016%, except for oleic acid with relative affinity of < 0.0005%. Saturated NEFAs, lauric, myristic, palmitic, and stearic acid were inactive. After purification by high-performance liquid chromatography, 1-oleoyl and 2-oleoyl lysolecithin displaced [125I]T4 from TBG with an affinity of 0.0006 and 0.0005%, respectively. On a molar basis, this affinity was approximately 10-fold lower than arachidonic acid, the most potent NEFA in inhibiting T4 binding to TBG in this assay system. Of all the NEFAs tested, only arachidonic acid inhibited [125I]T4 binding to TTR, with an affinity relative to unlabeled T4 of 0.49%. 1-Oleoyl, 1-palmitoyl, and 1-stearoyl lysolecithin were without effect on TTR binding. The T4-displacing effects of NEFAs are markedly attenuated by their extensive binding to albumin. Using purified [14C]NEFA preparations and heptane partitioning, the mean unbound percentages of linoleic, eicosapentaenoic, and docosahexaenoic acid in undiluted normal human serum were 0.00099, 0.0050, and 0.0042%, respectively (n = 3). In view of the very high degree of albumin binding of NEFAs, studies in diluted serum will grossly overestimate their competitor potency. The affinities of lysolecithins for the T4 binding sites of TBG and TTR are lower than those of NEFAs and depend on the fatty acid component. Lysolecithins are unlikely to influence plasma protein binding of T4 during critical illness.

Down-regulation of thyroxine-binding globulin messenger ribonucleic acid by 3,5,3'-triiodothyronine in human hepatoblastoma cells.

A sensitive [125I]-T4 binding assay was used to measure serum T4-binding globulin (TBG) in 60 individuals selected on the basis of their total circulating T3 concentrations, and a relationship between TBG and circulating thyroid hormone levels in humans was confirmed. There was a significant correlation between serum TBG and T3 or free T4 index. TBG secretion and TBG messenger ribonucleic acid (mRNA) production were studied with a continuous culture of the human hepatoblastoma cell line, HepG2. Cells were maintained in serum-free media for experimental manipulations. The addition of 100 nmol/L T3 to the cell medium resulted in a time-dependent down-regulation of TBG mRNA to 33 +/- 6% (+/- SD, n = 4) of untreated control levels by 24 h. Suppression of TBG mRNA was first detectable at 8 h (57% of untreated control levels). The effect of T3 was dose-responsive, with half-maximal suppression of TBG mRNA occurring at a bioavailable T3 concentration of approximately 30 pmol/L. The effect of T3 on TBG mRNA was not caused by a change in mRNA stability. Proteins secreted by HepG2 cells bound T4 with an affinity identical to that of normal circulating TBG. Cell secretion of TBG was parallel to total protein secretion and consistent with a TBG secretion rate of 50 ng/10(6) cells per day. Variations in the concentration of secreted binding protein in the presence of T3 corresponded to the changes observed in TBG mRNA. These data show that circulating TBG concentration is negatively correlated with total serum T3 in vivo. The corresponding down-regulation observed between TBG mRNA and secreted protein in HepG2 cells suggests that this effect is the result of the action of T3 on cellular TBG mRNA synthesis.

Alterations in hepatocyte uptake and plasma binding of thyroxine in nonthyroidal illness and caloric deprivation.

Low plasma T(3) in severe illness is widely thought to be due principally to inhibition of 5'-deiodinase activity, but other factors also contribute to this response. Abnormal plasma constituents, namely, 3-carboxy-4-methyl-5-propyl-2-furan propanoic acid (CMPF) and indoxyl sulfate in uremia, and elevated bilirubin and nonesterified fatty acids (NEFA) can impair T(4) transport into hepatocytes, thereby contributing to the lowering of plasma T(3). Assessment of possible endogenous or exogenous inhibitors of T(4) binding to plasma proteins is prone to dilution-dependent artifacts, which can lead to overestimation or underestimation of competitor potency, depending on experimental details. Because the potency of such competitors is a function of their free concentrations in undiluted serum, inhibitory activity may be enhanced by substances that impair their albumin binding. Oleic acid or CMPF can inhibit the effect of drugs such as furosemide or fenclofenac.