Synthesis and characterization of novel natural product-Gd(III) MRI contrast agent conjugates.

Several novel gadolinium chelates conjugated with paclitaxel, colchicine and thyroxine have been prepared as MRI contrast agents targeted to tubulin and thyroxine-binding globulin, respectively.

Characterization of T(4)-binding globulin cleaved by human leukocyte elastase.

T(4)-binding globulin (TBG) serves to maintain an important serum pool of thyroid hormones and to prevent their excessive loss in urine. TBG has also been implicated in the tissue distribution and targeted delivery of the hormones, the mechanisms of which remain unclear. By virtue of sequence homology, TBG belongs to the serine proteinase inhibitors superfamily of proteins that are characterized by a reactive site loop serving as a recognition site for serine proteinases. However, both TBG and another serpin with hormone transport function, corticosteroid-binding globulin, are noninhibitory. Cleavage of corticosteroid-binding globulin by human leukocyte elastase results in the reduction of its hormone-binding affinity and capacity. In this communication we confirm previous observations that TBG is also cleaved by elastase and undergoes the characteristic conformational changes. In addition, contrary to a previous report, the present work demonstrates that the cleaved product has reduced T(4)-binding affinity and, as expected, increased heat stability. Additional fragmentation of the molecule results in the loss of the hormone-binding site that is in agreement with a recent in vivo observation of apparent consumption at sites of inflammation. These data suggest that TBG may play a role in the targeted delivery of thyroid hormones to tissues rich in proteinases.

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.

Potential mechanisms of thyroid disruption in humans: interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin.

Organochlorine compounds, particularly polychlorinated biphenyls (PCBs), alter serum thyroid hormone levels in humans. Hydroxylated organochlorines have relatively high affinities for the serum transport protein transthyretin, but the ability of these compounds to interact with the human thyroid receptor is unknown. Using a baculovirus expression system in insect cells (Sf9 cells), we produced recombinant human thyroid receptor ss (hTRss). In competitive binding experiments, the recombinant receptor had the expected relative affinity for thyroid hormones and their analogs. In competitive inhibition experiments with PCBs, hydroxylated PCBs (OH-PCBs), DDT and its metabolites, and several organochlorine herbicides, only the OH-PCBs competed for binding. The affinity of hTRss for OH-PCBs was 10,000-fold lower (Ki = 20-50 microM) than its affinity for thyroid hormone (3,3',5-triiodothyronine, T3; Ki = 10 nM). Because their relative affinity for the receptor was low, we tested the ability of OH-PCBs to interact with the serum transport proteins--transthyretin and thyroid-binding globulin (TBG). With the exception of one compound, the OH-PCBs had the same affinity (Ki = 10-80 nM) for transthyretin as thyroid hormone (thyroxine; T4). Only two of the OH-PCBs bound TBG (Ki = 3-7 microM), but with a 100-fold lower affinity than T4. Hydroxylated PCBs have relatively low affinities for the human thyroid receptor in vitro, but they have a thyroid hormonelike affinity for the serum transport protein transthyretin. Based on these results, OH-PCBs in vivo are more likely to compete for binding to serum transport proteins than for binding to the thyroid receptor.

Nicotinic acid decreases serum thyroid hormone levels while maintaining a euthyroid state.

OBJECTIVE: To evaluate the effects of nicotinic acid on serum thyroid hormone levels in the absence of systemic illness or hepatic dysfunction. DESIGN: We determined the effect of treatment with nicotinic acid on serum thyroid hormone levels in one female and four male patients (mean age, 44.4 years) with hyperlipidemia. MATERIAL AND METHODS: In the five study patients, we measured serum lipids in conjunction with serum thyroxine (T4), triiodothyronine (T3) resin uptake, T3, free T4, thyroid-stimulating hormone (TSH), and thyroxine-binding globulin before, during, and after treatment with nicotinic acid. RESULTS: Serum lipid levels responded appropriately to nicotinic acid treatment. Thyroid function studies done a mean of 1.3 years (range, 0.5 to 3.5) after initiation of nicotinic acid therapy (mean daily dose, 2.6 +/- 0.7 g) revealed significant decreases in serum levels of total T4 (21%), free T4 index (16%), T3 (13%), and thyroxine-binding globulin (23%) (P < 0.02), whereas no significant changes were noted in free T4, T3 resin uptake, and TSH levels. During the course of treatment, the patients, who were carefully questioned, had no symptoms of hypothyroidism. Hypothyroidism was further excluded in three patients who had a normal serum TSH response to administration of thyrotropin-releasing hormone. In two patients, measurements of thyroid function returned to pretreatment levels after discontinuation of nicotinic acid therapy. No patient had significant abnormalities in liver-associated enzymes or evidence of systemic illness during the course of treatment. CONCLUSION: These results suggest that nicotinic acid decreases serum thyroid hormone concentrations while maintaining a euthyroid state. This effect may be mediated through reduction in thyroxine-binding globulin, but other mechanisms may also be involved.

Effect of four different oral contraceptives on various sex hormones and serum-binding globulins.

In a double-blind, controlled, randomized, four-arm, bicentric clinical study, the effect of four oral contraceptives (OCs) on various hormone parameters and serum-binding globulins was investigated. Four groups with 25 volunteers each (18-35 years of age) were treated for six cycles with monophasic combinations containing 21 tablets with either 30 microg ethinylestradiol (EE) + 2 mg dienogest (DNG) (30EE/DNG), 20 microg EE + 2 mg DNG (20EE/DNG), 10 microg EE + 2 mg estradiol valerate (EV) + 2 mg DNG (EE/EV/DNG) or 20 microg EE + 100 microg levonorgestrel (LNG) (EE/LNG). The study was completed by 91 subjects. Blood samples were taken after at least 12 h of fasting on Day 21-26 of the preceding control cycle and on Day 18-21 of the first, third and sixth treatment cycle. The serum concentrations of free testosterone were significantly decreased by about 40-60% in all four groups, while those of dehydroepiandrosterone sulfate (DHEAS) showed a time-dependent decrease during treatment. Except for EE/EV/DNG, which increased prolactin significantly during the third and sixth cycles, no change was observed with the EE-containing preparations. There was a significant increase in the levels of serum-binding globulins during treatment, which differed according to the composition of the OCs used. The rise in sex hormone-binding globulin (SHBG) was highest during intake of 30EE/DNG (+320%) and lowest with EE/LNG (+80%), while the effect of 20EE/DNG and EE/EV/DNG was similar (+270%). The thyroxine-binding globulin (TBG) levels increased significantly, by 50-60%, during treatment with the DNG-containing formulations, while the effect of EE/LNG was less significant (+30%). The rise in corticosteroid-binding globulin (CBG), which occurred in all groups, was most pronounced in women treated with 30EE/DNG (+90%) and least with EE/EV/DNG (+55%), indicating a strong influence of EE and no effect of the progestogen component. In all treatment groups, the frequency of intracyclic bleeding rose in the first treatment cycle and decreased thereafter. Cycle control was significantly better with 30EE/DNG or EE/LNG than with 20EE/DNG or EE/EV/DNG. There was no significant change in blood pressure, body mass index or pulse rate throughout the study. In conclusion, the DNG-containing OCs caused a higher rise in SHBG and TBG levels than the LNG-containing preparation. The effects on CBG suggest a lesser hepatic effect of 2 mg EV as compared to 20 or 30 microg EE. In contrast to EE, the use of estradiol in OCs appeared to increase prolactin release, while the cycle control was better with the OC containing 30 microg EE.

A case of pseudo-Bartter's syndrome associated with hypergastrinemia, thrombocytosis and increased serum thyroxine-binding globulin.

A housewife, 40 years of age, was admitted with dysesthesia of the extremities, muscle weakness, and attacks of adynamia and thirst. She had been taking a laxative for more than 20 years. On physical examination, blood pressure was 94/56 mmHg. Laboratory tests revealed thrombocytosis, low serum K and marked increases in both plasma renin activity and serum aldosterone. Serum TBG was increased. Serum gastrin was also markedly increased and could not be enhanced by exogenous secretin. Both angiotensin 11 loading test and noradrenalin loading test failed to increase blood pressure. Ammonium chloride loading to examine the disturbance of urinary acidification was abnormal in the short term test and borderline in the long term test. Following a diagnosis of pseudo-Bartter's syndrome induced by long term intake of laxative and repeated diarrhea, the administration of laxative was interrupted and potassium, indomethacin and spironolactone were administered. However, serum K remained low. Hypergastrinemia, thrombocytosis and a high serum TBG level also persisted, the causes of which remain unknown. This is the first reported case of pseudo-Bartter's syndrome associated with hypergastrinemia, thrombocytosis and increased serum TBG.

Effects of short-term 17 beta-estradiol treatment on the properties of T4-binding proteins in the plasma of immature rainbow trout, Oncorhynchus mykiss.

To determine the effects of 17 beta-estradiol (E2) on the properties of the plasma proteins that bind L-thyroxine (T4) immature rainbow trout, Oncorhynchus mykiss, were injected intraperitoneally on days 0 and 3 with 0.5 mg E2-3-benzoate/100 g body weight, and plasma was sampled on days 4, 7, or 12. Control trout received peanut oil alone. E2 caused a small but significant decrease in the free T4 index. Saturation analysis on miniature G-25 Sephadex columns revealed at least two major T4-binding sites. Filtration on agarose Bio-gel A 1.5 also indicated two major T4-binding protein fractions with molecular weights of 150 and 55 kDa with a small proportion of T4 binding to a 1,500-kDa site presumed to be lipoprotein. Addition of unlabeled T4 displaced [125I]T4 from the 55-kDa site and unmasked an adjacent site of higher molecular weight. E2 increased the proportion of T4 bound to the low-affinity (150 kDa) site relative to that bound to the high-affinity (55 kDa) site, increased the level of protein associated with the 1,500-kDa site and its T4 binding, and also initiated the production of presumed vitellogenin (VTG), which bound a small amount of T4. It is concluded that the E2-induced depression in FT4 is caused by a shift in T4 binding between high-affinity and low-affinity sites, and also by binding of small amounts of T4 to presumed lipoprotein and VTG.

Alterations in serum thyroid hormonal indices with colestipol-niacin therapy.

A serial blood-lipid-lowering study at the University of Southern California yielded unexpected findings on routine thyroid function monitoring. After 1 year of combined colestipol and niacin therapy, patients had reduced total serum thyroxine (T4) levels and increased triiodothyronine uptake ratios, an indicator of apparent decreases in thyroxine-binding globulin levels. Calculation of the free T4 index partially but not completely corrected for the apparent decrease in thyroxine-binding globulin, as determined by a relatively small decrease in the free T4 index compared with a large decrease in T4. Sequential sampling, using three separate methods, showed reduced thyroxine-binding globulin levels. The mechanism for these changes is unknown, but the fact that these patients were essentially euthyroid needs emphasis because the use of combined colestipol and niacin therapy is becoming more widespread.

Decreased levothyroxine requirement in women with hypothyroidism during androgen therapy for breast cancer.

OBJECTIVE: To determine the effects of androgen administration on measures of thyroid function and thyroid hormone replacement doses in women with breast cancer. DESIGN: Consecutive patients with metastatic, hormone-dependent breast cancer who were eligible for androgen treatment. INTERVENTIONS: Androgen therapy (fluoxymesterone, 10 mg orally twice daily) was continued for as long as it was effective in controlling tumor growth. PATIENTS: 7 patients with no known thyroid disease and 4 others receiving long-term treatment for hypothyroidism. MEASUREMENTS: Serum levels of total and free thyroxine (T4), thyroid-stimulating hormone (TSH), and T4-binding globulin were determined before and every 4 weeks after androgen therapy was initiated. RESULTS: Within 4 weeks of androgen administration to the seven patients without thyroid disease, serum levels of total T4 and T4-binding globulin decreased (P < 0.001), whereas the calculated free thyroxine index and measured free hormone levels remained unchanged. Six to 12 weeks after androgen therapy was discontinued, all seven patients remained clinically euthyroid, and serum levels returned to baseline values. In contrast, clinical hyperthyroidism developed shortly after androgen was administered to four patients who received long-term thyroid hormone replacement therapy. Within 4 weeks of treatment, the serum free T4 level increased in each of the four patients, whereas the TSH level decreased. Thyroid hormone doses had to be reduced by 25% to 50% to maintain euthyroidism. CONCLUSIONS: The study documents the reversible effects of androgens on thyroid hormone levels and indicates the need to reduce thyroid replacement doses in women during androgen therapy. Monitoring thyroid hormone levels in patients receiving replacement therapy and perhaps in those with autonomous thyroid function is necessary after androgen therapy.

Thyroid function in postmenopausal breast cancer patients treated with tamoxifen.

Thyroid function was evaluation in 26 postmenopausal women with breast cancer before and at various time intervals during treatment with tamoxifen. Tamoxifen treatment suppressed plasma levels of FT3 and FT4 (p < 0.005 for both) and elevated plasma concentrations of TBG (p < 0.005 and TG (p < 0.025). In general, these changes became significant after 6 months of treatment. Plasma TSH increased significantly after 1 y of treatment (p < 0.025). A fall in FT4 and FT3 combined with increase in TSH suggests a reduced bioavailability of T4 and T3 during tamoxifen treatment. The increase in TG may reflect a reduced synthesis or liberation of T4 resulting in a reduced plasma level of FT4. Our findings suggest that tamoxifen influences the thyroid hormone levels, not only by modulating plasma TBG, but also by interfering with hormone synthesis or secretion in the thyroid gland.