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.

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.

Segmental renin sampling and partial nephrectomy in renal hypertension.

Selective renin sampling from renal vein tributaries identified a high-renin source in the lower pole of the left kidney in a 16-year-old boy who had gradually developed hypertension after blunt left renal trauma. Localized renin secretion from the ischemic pole was associated with suppression of renin secretion from both the contralateral kidney and the normal part of the affected kidney. Removal of ischemic tissue by partial nephrectomy produced sustained correction of hypertension. The findings indicate that segmental renin sampling can define indications for partial nephrectomy in renal hypertension.

Transient anterior electrocardiographic changes simulating acute anterior myocardial infarction in diabetic ketoacidosis.

In two patients with severe diabetic ketoacidosis, electrocardiography showed transient anterior changes suggestive of acute transmural infarction without subsequent evidence of myocardial necrosis. While the mechanism of these and other temporary electrocardiographic changes in diabetic ketoacidosis remains unclear, appreciation of their transient nature is essential if misdiagnosis of myocardial infarction and possible inappropriate delay in intravenous fluid administration are to be avoided. When electrocardiographic abnormalities are present early in diabetic ketoacidosis, the full 12-lead electrocardiogram should be repeated after adequate resuscitation.

19-nor analogs of adrenal steroids: mineralocorticoid and glucocorticoid receptor activity.

The effect of absence of the C-19 methyl group from five adrenal steroids has been studied in terms of their affinity for mineralocorticoid (MR) and glucocorticoid receptors (GR). In MR assays, 19-nordeoxycorticosterone and 19-norprogesterone showed 3-fold higher affinity for MR than did their respective parent steroids; 19-norcortisol had 1.5 times the affinity of cortisol for MR. In contrast, corticosterone and 19-nororticosterone showed equal affinity, and 19-noraldosterone showed less than 1% the MR activity of aldosterone. In GR assays, the absence of the C-19 methyl group from progesterone increased GR affinity 3-fold and deoxycorticosterone affinity 1.5-fold. In contrast, the other 19-nor steroids showed decreased affinity vis à vis their parent compounds (19-norcorticosterone, 30%; 19-norcortisol, 10%; 19-noraldosterone, < 1%). These findings suggest that while the 19-nor analogs of 11-deoxy steroids are consistently more active than their parent steroid, the 19-nor 11-oxygenated adrenal steroids show no predictable pattern of binding for MR or GR.

Uptake of 3,5,3'-triiodothyronine by cultured rat hepatoma cells is inhibitable by nonbile acid cholephils, diphenylhydantoin, and nonsteroidal antiinflammatory drugs.

Cellular uptake of T3 was examined using rat H4 hepatoma cells. Uptake of [125I]T3 (10(-11) M) from serum-free medium was measured as the cell-associated counts retained by washed cells (2 X 10(6) per well). Displaceable uptake was 84% of total uptake at 2 min (2.9% of total counts). T4, tetraiodothyroacetic acid, triiodothyroacetic acid, rT3, and D-T3 were 2-5% as effective as T3 in displacing uptake. Nonequilibrium kinetics indicated a half-maximal uptake at 680 nM T3 with approximately 7 million sites per cell. Displaceable uptake was time and temperature dependent and was 73% inhibited by 2 mM KCN and 52% by 10 mM bacitracin but not by 2 mM ouabain or 10 microM cytochalasin B. Phloretin, 100 microM, inhibited uptake by 66%. T3 uptake was directly related to the free T3 concentration over the range of albumin concentrations, 0-10 g/liter. The nonbile acid cholephil compounds, bromosulfophthalein, iopanoic acid, and indocyanine green (all 100 microM) inhibited T3 uptake to 62%, 17%, and 5% of control, respectively. Taurocholate, methylaminoisobutyric acid, and oleic acid were noninhibitory. The half-inhibitory concentrations of reactive nonsteroidal antiinflammatory drugs were: meclofenamic acid (25 microM), mefenamic acid (45 microM), fenclofenac (69 microM), flufenamic acid (100 microM), and diclofenac (230 microM). Aspirin, ibuprofen, oxyphenbutazone, and phenylbutazone (all 100 microM) were noninhibitory. Diphenylhydantoin inhibited uptake to 50% at 75 microM. These findings suggest that T3 uptake by cultured rat hepatocytes is by an energy-dependent, saturable, stereo-selective mechanism that is dependent on cell membrane proteins. This mechanism appears to be shared by a number of other ligands, including nonbile acid cholephils and several nonsteroidal antiinflammatory drugs of the anthranilic and phenylacetic acid classes, as well as diphenylhydantoin. The bile acid taurocholate, oleic acid, and a probe for type A amino acid uptake were inactive. The extent to which these effects may modify expression of thyroid hormone action remains to be established.

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.

Long term evolution of glucocorticoid-suppressible hyperaldosteronism.

It is generally held that idiopathic hyperaldosteronism and glucocorticoid-suppressible hyperaldosteronism (GSH) are distinct entities, distinguishable by thorough investigation. We report a patient who presented in 1974 at age 15 yr with blood pressure of 240/120 mm Hg, serum K of 3.1 mM, low renin, and high normal aldosterone excretion, with findings diagnostic of GSH. After dexamethasone treatment (2 mg/day for 4 weeks), urinary aldosterone was undetectable (less than 1 microgram/24 h), associated with correction of hypertension and hypokalaemia. While untreated, plasma aldosterone increased in response to ACTH infusion at a dose that did not influence plasma cortisol. Plasma aldosterone at 0800 h while recumbent was higher than in subsequent samples taken while ambulant, consistent with ACTH dependence of aldosterone secretion. Blood pressure, renin, and potassium remained normal for 4 yr during treatment with dexamethasone (0.5-0.75 mg/day), but hypertension then slowly returned. After 7 yr, the original studies were repeated. Urinary aldosterone excretion was again in the high normal range after 3 weeks of no treatment, but both plasma renin and aldosterone consistently increased in response to upright posture. After dexamethasone treatment (2 mg/day) for 4 weeks, urinary aldosterone was not suppressed (excretion rate, 10.8, 9.2, and 5.7 micrograms/day; urinary Na, greater than 100 mmol/day; urinary cortisol, less than 1 microgram/day). At this time, dexamethasone did not alleviate hypertension or increase renin. Control of blood pressure has been readily achieved, 8-12 yr after diagnosis, with a low dose of beta-adrenergic antagonist and amiloride. Aldosterone remains incompletely suppressible during sodium loading, so that the findings now resemble those of idiopathic hyperaldosteronism. This sequence indicates that glucocorticoids may not permanently control hypertension in GSH. The transient dominance of ACTH in control of aldosterone secretion indicates that the relationship between GSH and idiopathic hyperaldosteronism merits further evaluation in long term studies.

Hyperthyroidism without triiodothyronine excess: an effect of severe non-thyroidal illness.

Serial changes in thyroid hormone levels are described in two patients in whom hyperthyroidism was associated with transient non-thyroidal illness. In a 74-year-old woman with mild hyperthyroidism, two episodes of cholecystitis were associated with subnormal concentrations of serum T3 and increased concentrations of serum rT3; T3 became elevated during recovery, associated with a simultaneous fall in rT3. The TSH response to TRH was undetectable on three occasions. A cholecystectomy was performed after preparation with Lugol's iodine and subsequent tests showed evolution through T3 toxicosis to classical hyperthyroidism. In the second case, symptoms and signs of classical hyperthyroidism were noted during an undiagnosed illness characterized by severe abdominal pain and fever. Six days after the onset of this illness, an elevated level of serum T4 was associated with a normal total T3 concentration and increased concentration of rT3. After resolution of abdominal symptoms, serum T3 was markedly increased, associated with persistent T4 and rT3 excess. These findings indicate that the changes in T3 and reverse T3 described in non-thyroidal illness also occur in hyperthyroid patients, and suggest that the fall in T3 may be of sufficient magnitude to make T3 measurement diagnostically unreliable in the presence of non-thyroidal illness.

Interactions between oleic acid and drug competitors influence specific binding of thyroxine in serum.

Long chain nonesterified fatty acids and various drugs may share albumin-binding sites in common. We questioned whether serum binding of T4 could be indirectly influenced by displacement of drug competitors from these sites by nonesterified fatty acids. The influence of oleic acid on drug-induced inhibition of [125I]T4 binding was measured by equilibrium dialysis, using undiluted serum in order to avoid dilution-related artefacts. Oleic acid (1 mmol/L) alone did not inhibit serum protein binding of T4, but this concentration augmented the inhibitory effects on T4 binding of diflunisal, mefenamic acid, meclofenamic acid, and aspirin. This effect increased with increasing concentrations of mefenamic acid, meclofenamic acid, and furosemide. The T4-displacing effect of fenclofenac was not augmented by oleic acid. The mechanism of these interactions was studied by examining 1) oleic acid effects on drug binding, and 2) drug effects on oleic acid binding in undiluted serum. Increments in added oleic acid (0.5-2.0 mmol/L) progressively increased the mean unbound fractions of [14C]aspirin, [14C] diflunisal, and [14C]furosemide, but did not displace [14C]fenclofenac. At the relevant total and free drug concentrations, the inhibitory effect of oleic acid on drug binding and its influence on drug-induced displacement of T4 were concordant in the order: meclofenamic acid greater than aspirin greater than mefenamic acid greater than diflunisal greater than furosemide greater than fenclofenac. In contrast, drug-induced increases in the unbound fraction of [14C]oleic acid did not correlate with augmentation of T4 displacement. We conclude that synergistic effects of oleic acid and drugs on T4 binding result from drug displacement by oleic acid, rather than the reverse effect. Hence, substances that increase the unbound concentration of a competitor by displacing it from albumin can increase its T4-displacing potency. Interactions between various ligands may exert a greater hormone-displacing effect than the sum of each alone.

Specific methods to identify plasma binding abnormalities in euthyroid hyperthyroxinemia.

Methods to identify the plasma T4-binding abnormalities that can cause euthyroid hyperthyroxinemia were evaluated in patients with excess T4-binding globulin, familial dysalbuminemic hyperthyroxinemia, prealbumin-associated hyperthyroxinemia, and autoantibody binding of T4. Familial dysalbuminemic hyperthyroxinemic serum showed a unique persistence of abnormal [125I]T4 binding when diluted 1:100 in phosphate buffer with added 1000-fold excess of unlabeled T4 (10(-6) M T4). Immunoprecipitation of [125I]T4 by antibody to prealbumin, precipitation of [125I]T4 by polyethylene glycol 6000 19%, and in vitro resin uptake of T3 were specific for prealbumin-associated hyperthyroxinemia, autoantibody binding of T4, and T4-binding globulin excess, respectively. These simple methods facilitate investigation of patients with euthyroid hyperthyroxinemia and will identify individuals and families at risk of misdiagnosis by standard methods. Use of these techniques rules out the known binding abnormalities in hyperthyroxinemic patients and may make the diagnosis of generalized hormone resistance more specific.

Familial euthyroid thyroxine excess: characterization of abnormal intermediate affinity thyroxine binding to albumin.

The abnormal high capacity T4 binding site of familial euthyroid T4 excess was separable from prealbumin and T4-binding globulin but not from albumin. We therefore compared T4 binding by albumin preparations isolated from the sera of normal and affected subjects. By equilibrium dialysis, albumin from affected subjects showed an extra T4 binding site (Kd approximately 50 nM) in addition to the T4 binding sites of normal albumin (Kd approximately 4 microM). Comparison of the estimated capacity of the additional site (200 microM) with the molar concentration of albumin suggested that only about one third of albumin molecules from affected subjects contained the extra binding site. Estimates of affinity and capacity were used to derive combining powers for the diverse classes of serum T4 binding sites. From these estimates, it appears that the presence of the abnormal site accounts for the approximate doubling of normal mean total T4 (from approximately 100 nM or 7.7 micrograms/dl to approximately 200 nM or 15.5 micrograms/dl), in order to maintain a normal free T4 in the face of the increased T4 association with albumin. Studies of [125I]T4 displacement from albumin of affected subjects showed low T3 affinity and competition by barbitone. Relative molar concentrations to give equivalent displacement of [125I]T4 were: 3,3',5,5'-tetraiodothyroacetic acid, 0.4; T4, 1.0; rT3, 4; 8-anilinonaphthalene sulfonic acid, 10; T3, 80; salicylate, 200; and barbitone, 40,000. Studies with dithiothreitol suggested that disulfide bonds were critical in maintaining the T4-albumin association. These findings indicate that familial T4 excess is due to abnormal intermediate affinity, sulfhydryl-sensitive T4 binding sites that are inseparable from the albumin found in affected subjects.

Visual failure during replacement therapy in primary hypothyroidism with pituitary enlargement.

A 34-year-old woman with longstanding untreated thyroprivic hypothyroidism and pituitary enlargement is reported here in whom visual failure coincided with thyroid hormone replacement. Visual fields were normal after 30 years untreated hypothyroidism, but severe concentric field constriction developed during the first 6 months of therapy and was relieved by hypophysectomy. Plasma TSH and prolactin remained elevated during 10 months replacement therapy, but both were suppressed by preoperative hyperreplacement with T3 and T4. The paradoxical pressure symptoms suggest imbalance between pituitary TSH content and TSH release during treatment with thyroid hormone; a finding previously reported in animal studies. This sequence suggests that patients with known pituitary enlargement secondary to thyroid hypofunction should be observed for pressure symptoms during thyroid hormone treatment.

Drug and fatty acid effects on serum thyroid hormone binding.

We directly compared the competitor potency for serum T4 binding of 11 nonsteroidal antiinflammatory drugs; the diuretics furosemide, ethacrynic acid, and bumetanide; diphenylhydantoin; the cholecystographic contrast agents iopanoate and ipodate; and six long-chain nonesterified fatty acids (NEFA) using equilibrium dialysis. To avoid artefacts that occur in competitor studies with diluted serum or isolated binding proteins, we used undiluted normal serum, with drugs added at concentrations that achieved high therapeutic total and free serum levels at equilibrium. Drug addition was based on the measured free fraction of each drug in serum. The free T4 fraction in normal serum (Tris buffer, pH 7.4; 37 C) was between 1.40 X 10(-4) and 1.53 X 10(-4). Drug-induced increases in T4 free fraction were: fenclofenac, 90%; aspirin, 62%; meclofenamic acid, 39%; diflunisal, 37%; mefenamic acid, 31%; and furosemide, 31%. Significant increases of 7-15% occurred with diclofenac, flufenamic acid, phenylbutazone, and diphenylhydantoin. Indomethacin, ketoprofen, tolmetin, ethacrynic acid, bumetanide, iopanoate, and ipodate were inactive at the concentrations studied. Addition of 2.0 mmol/L oleic acid had a negligible effect, but 3.5 mmol/L oleic acid inhibited T3 and T4 binding significantly. Other long chain NEFA (addition of 1.5 mmol/L) gave increases in free T4 fraction as follows: arachidonic acid, 26%; linolenic acid, 23%; and linoleic acid, 11%. Stearic and palmitic acids were inactive. The effect of 5 mmol/L oleic acid in serum could be reproduced by addition of 0.5 mmol/L to serum diluted 1:10, indicating that protein binding of NEFA is the major determinant that limits their competitor potency. These findings provide a basis for anticipating which potential inhibitors may cause important changes in serum thyroid hormone binding. The time course of such effects will be influenced by the pharmacokinetics of the inhibitor itself as well as the equilibrium findings described here.

Familial euthyroid thyroxine excess: an appropriate response to abnormal thyroxine binding associated with albumin.

Responses of the pituitary-thyroid axis and T4 binding to plasma proteins were studied in three kindreds with familial euthyroid T4 excess, an autosomal dominant condition in which affected subjects have high concentrations of plasma T4 with a high free T4 index, but normal free T4 by equilibrium dialysis. Treatment of affected subjects with exogenous T4 or T3 led to gradual suppression of TSH secretion when the free level of T4 or T3 increased above normal. When total T4 was reduced toward normal by potassium iodide treatment or previous subtotal thyroidectomy, the findings suggested mild hormone deficiency. In affected subjects from all three families, equilibrium dialysis showed increased [125I]T4 binding, with evidence of abnormal high capacity binding when an excess of unlabeled T4 was added. In contrast, T3 binding showed no major abnormality. Serum concentrations of T4-binding globulin, prealbumin, and albumin were normal, but gel electrophoresis and immunoprecipitation of binding proteins indicated that 25-30% of tracer [125I]T4 was albumin bound (normal, 10-12%). Abnormal binding, studied by an adsorption separation system in the presence of T4 excess, was inhibited by increments of barbitone. These findings suggest that T4 excess is an appropriate response to abnormal T4 binding so as to maintain normal free T4. The excess bound T4 is associated with a normal quantity of albumin. The basis for increased T4-albumin binding remains to be determined.

The effect of triamterene and sodium intake on renin, aldosterone, and erythrocyte sodium transport in Liddle's syndrome.

Liddle's syndrome was diagnosed in a 23-yr-old Chinese girl with hypertension and hypokalemia by the presence of suppressed renin and negligible plasma and urinary aldosterone secretion. Adrenal corticosteroids, including aldosterone, were suppressed by dexamethasone and stimulated by ACTH. While spironolactone was ineffective, triamterene (2,4,7-triamino-6-phenyl-pteridine) treatment corrected the hypertension and hypokalemia and restored PRA to normal provided that sodium intake was not excessive. During long term treatment with triamterene, blood pressure was extremely sensitive to salt intake, increasing promptly with high intake and decreasing with low salt intake. As a result of the chronic hypervolemia and sodium retention consequent upon the patient's persistent high salt intake and increased renal tubular sodium reabsorption, plasma renin and aldosterone remained low. Erythrocyte sodium concentration and membrane permeability were increased. Triamterene with salt restriction was able to lower the intracellular sodium concentration but did not correct the increased sodium permeability. This suggests that there is an abnormality of sodium transport in Liddle's syndrome which affects the erythrocytes as well as the renal tubular cells.

Thyroxine binding by human transthyretin variants: mutations at position 119, but not position 54, increase thyroxine binding affinity.

A mutation at codon 119 in the transthyretin (TTR) gene leads to a substitution of methionine for threonine at this position in the circulating protein. As the amino acid at position 119 is located in the T4 binding channel, mutations here may affect the binding of T4 by TTR. A previous study has shown an increase in the amount of hormone carried by the TTRMet119 variant. To determine whether this increase in binding was due to a change in affinity or capacity, TTR was partially purified from normal individuals and those with the TTRMet119 mutation. The isolation procedure was a rapid, single step passage through Blue Sepharose. With normal serum, the resulting protein bound T4 with a single site of intermediate affinity (Ka, 1.63 +/- 0.36 x 10(7) L/mol). No sites of higher or lower affinity were detected. Comparisons of binding capacity and immunoreactive TTR concentrations showed that the preparations bound T4 with a molar ratio between 1-2. With TTRMet119 serum, the T4 affinity was approximately doubled [Ka, 3.40 +/- 0.76 x 10(7) L/mol (+/- SD); P < 0.001] with no change in binding capacity. This doubling in affinity explains the observed T4 levels of about 120 nmol/L in individuals with this mutation. Binding of rT3 to TTRMet119 was increased approximately 5-fold over normal. Identical experiments with TTRGly54, in which glycine is substituted for glutamine, showed that the T4 affinity of this variant was unchanged from normal. These results suggest that the TTRMet119 mutation leads to secretion of a normal concentration of TTR that has a raised affinity for T4. Depending on their location, mutations in the TTR gene may lead to an increase or no change in T4 binding by the secreted protein.

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.

Interaction of furosemide with serum thyroxine-binding sites: in vivo and in vitro studies and comparison with other inhibitors.

The diuretic furosemide inhibits serum protein binding of T4 in equilibrium dialysis, dextran-charcoal, and competitive ligand binding separation systems and displaces [125I]T4 from isolated preparations of T4-binding globulin (TBG), prealbumin, and albumin. Equilibrium dialysis studies of undiluted normal serum showed that about 10 micrograms/ml furosemide increased the free T4 and free T3 fractions. Displacement occurred at lower drug concentrations in sera with subnormal albumin and TBG levels. Binding of [14C]furosemide to TBG was inhibited by unlabeled T4, suggesting that furosemide and T4 share a common binding site. A single oral dose of 500 mg furosemide given to five patients maintained on peritoneal dialysis increased the percentage of charcoal uptake of [125I]T4 (using serum diluted 1:10) from 4.1 +/- 1.0 (+/- SE) to 10.8 +/- 4.3 (P less than 0.01) after 2 h, while decreasing total T3 from 75 +/- 5 to 56 +/- 13 ng/dl (P less than 0.01) and total T4 from 6.7 +/- 0.9 to 4.8 +/- 0.8 micrograms/dl (P less than 0.01) after 5 h. Various ligands inhibited [125I]T4 binding to serum proteins in the following relative molar relationship: T4, 1; furosemide, 1.5 X 10(3); fenclofenac, 2 X 10(4); mefenamic acid. 2.5 X 10(4); diphenylhydantoin, 4 X 10[4); ethacrynic acid, 10(5); heparin 5 X 10(5); 2-hydroxybenzoylglycine, 10(6); and sodium salicylate, 1.5 X 10(6). These studies demonstrate that furosemide competes for T4-binding sites on TBG, prealbumin, and albumin, so that a single high dose can acutely lower total T4 and T3 levels. The drug is much more potent on a molar basis than other drug inhibitors of T4 binding, but at normal therapeutic concentrations, furosemide is unlikely to decrease serum T4 or T3. However, high doses, diminished renal clearance, hypoalbuminemia, and low TBG accentuate its T4- and T3-lowering effect. Hence, furosemide should be considered a possible cause of low thyroid hormone levels in patients with critical illness. The significance of this drug in reports of impaired hormone and drug binding in renal failure requires further assessment.

Relationship between thyrotropin and thyroxine changes during recovery from severe hypothyroxinemia of critical illness.

In a prospective study of critically ill hypothyroxinemic we assessed the relationship between serum TSH and T4 during the return of serum T4 to normal during recovery. In this longitudinal study of 60 patients with a variety of critical illnesses, including burns, septicemia, and acute renal failure, serum T4 fell to less than 2.7 micrograms/dl (35 nmol/liter) in 24 patients, of whom 14 survived with return of T4 to normal. A rise in total T4 of more than 1.9 microgram/dl (25 nmol/liter) within 96 h occurred 13 times in 10 patients, while 4 patients had slower increases in T4. All 13 episodes of rapid T4 rise [1.7 +/- 0.8 (+/- SD) to 5.6 +/- 2.1 micrograms/dl] were associated with a marked increase in serum TSH (1.1 +/- 0.8 to 7.0 +/- 5.2 mU/liter), and TSH was transiently above normal during 8 episodes of T4 recovery. In the 6 episodes with sampling less than 6 h apart, the TSH rise consistently preceded the T4 rise. In the 4 patients who received dopamine, TSH and T4 remained low until cessation of therapy. During the TSH rise, only minor changes, which could not account for the increase in total T4, occurred in T4-binding globulin (12.9 +/- 3.3 to 14.8 +/- 3.3 mg/liter), prealbumin (208 +/- 73 to 234 +/- 82 mg/liter), and albumin (28.3 +/- 2.9 to 31.9 +/- 2.9 g/liter). Mean free T4 increased (0.60 +/- 0.34 to 1.45 +/- 0.56 ng/dl), as did total T3 (16 +/- 14 to 76 +/- 44 ng/dl), during the phase of TSH rise, suggesting that the increase in TSH was not simply a consequence of diminished negative feedback due to increased plasma binding. The very close and consistent temporal relationship between TSH and T4 during the recovery phase suggests that TSH may have an essential role in the return of T4 to normal during recovery from critical nonthyroidal illness.