Experimental studies of boron neutron capture therapy (BNCT) using histone deacetylase inhibitor (HDACI) sodium butyrate, as a complementary drug for the treatment of poorly differentiated thyroid cancer (PDTC).

PURPOSE: The present study analyzed different protocols of administration of boronophenylalanine (BPA) and sodium butyrate (NaB) to increase the BNCT efficacy for poorly differentiated thyroid cancer (PDTC). MATERIALS AND METHODS: Nude mice implanted with human PDTC cells (WRO) were distributed into four protocols: 1) BPA; 2) BPA + ip NaB; 3) BPA + oral NaB; 4) Control. Biodistribution and histologic studies were performed. LAT (BPA transporter) isoforms gene expression was assessed by RT-PCR. RESULTS: Tumor growth delay was observed in animals of the Protocol #3 (p < 0.05). NaB (Protocol #2) increased tumor boron uptake 2-h post BPA injection (p < 0.05). On the other hand, NaB upregulated the expression of all the isoforms of the LAT transporter in vitro. Histologic studies showed a significant decrease of mitotic activity and an increase of vacuoles in tumors of Protocol #3. Neutrons alone or combined with NaB caused some tumor growth delay (p < 0.05), while in the BNCT and BNCT + NaB groups, there was a halt in tumor growth in 70 and 80% of the animals, respectively. CONCLUSIONS: Intraperitoneally administration of NaB increased boron uptake while oral administration for a longer period of time induced tumor growth delay previous to BPA administration. The use of NaB via ip would optimize the irradiation results.

Inhibition of goiter growth and of cyclic AMP formation in rat thyroid by 2-iodohexadecanal.

INTRODUCTION: Thyroid autoregulation has been related to intraglandular content of an unknown putative iodocompound. The thyroid is capable of producing different iodolipids such as 6-iodo-deltalactone (ILdelta) and 2-iodohexadecanal (2-IHDA). Data from different laboratories have shown that these iodolipids inhibit several thyroid parameters. ILdelta has an antigoitrogenic action but no data about the action of 2-IHDA on this parameter has been published. OBJECTIVES: to study the action of 2-IHDA on methimazole (MMI)-induced goiter and analyze if this compound can cause the involution of preformed goiter. RESULTS: Administration of MMI to rats during 10 days increased thyroid weight by 112%. This effect was significantly inhibited by the simultaneous injection of 20mug/day of 2-IHDA (51% vs. MMI) while iodine or non iodinated hexadecanal were without action. Thyroidal proliferating cell nuclear antigen (PCNA) content was increased by MMI while 2-IHDA decreased this value (control: 100%; MMI: 190+/-11; MMI+2-IHDA: 134+/-10). Serum TSH was increased after MMI administration and 2-IHDA did not modify this parameter (control: 1.89+/-0.10; MMI: 8.19+/-0.93ng/ml; MMI+2-IHDA: 7.38+/-0.72). Treatment with MMI increased thyroidal cAMP content (control: 16.1+/-0.82, MMI: 42.4+/-4.6 fmol/mg protein) while injection of 2-IHDA significantly decreased this value (22.3+/-2.0). Goiter prevention by 2-IHDA was also observed at 30 days of treatment reducing total number of cells (51% inhibition) and epithelial height (81% inhibition). Goiter involution was induced after withdrawal of MMI and injection with 2-IHDA, KI or saline. 2-IHDA led to a reduction of 74.5% in thyroid weight after 3 days while spontaneous involution (saline) was only of 32%. KI failed to alter this value. This significant involution was accompanied by a reduction in the number of cells (66%). Administration of the iodolipids did not produce significant changes in several serum parameters such as total T(3) and T(4), cholesterol, transaminases, urea and creatinine. CONCLUSION: 2-Iodohexadecanal, as 6-iodo-deltalactone, prevents goiter growth in rats and opens a potential therapeutic application of iodolipids.

Inhibition of peroxidase and catalase activities and modulation of hydrogen peroxide level by inositol phosphoglycan-like compounds.

Inositol phosphoglycan-like compounds are produced by the hydrolysis of the membrane bound glycosyl phosphoinositides. Besides being short term mediators of insulin action, they inhibit peroxidases and catalase, increasing the concentration of cellular hydrogen peroxide. Although high concentrations of hydrogen peroxide are toxic, moderate increases of its basal level are signals for different metabolic pathways. The inhibitor, localized in the cytosol of the cell, acts on peroxidases and catalase of the same tissue (homologous action) and of other tissues or organisms (heterologous action). The inositol phosphoglycan-like compound inhibits peroxidases with different prosthetic groups, i.e. containing iron such as: thyroid peroxidase, lactoperoxidase, horseradish peroxidase, soy bean peroxidase; and containing selenium such as glutathione peroxidase and 2-cys peroxiredoxin with no prosthetic group. Besides peroxidases, the inositol phosphoglycan-like compound inhibits catalase, another heme enzyme. The inhibition kinetics demonstrates a noncompetitive effect. The site of action is not the prosthetic group, given that the inhibitor does not produce any effect on the peak in the Soret region in the presence or absence of hydrogen peroxide. In conclusion, the inositol phosphoglycan-like compound is the general inhibitor of peroxidases and catalase involved in the modulation of hydrogen peroxide level that acts in different metabolic pathways as a signal transducer.

Nicotinamide increases thyroid radiosensitivity by stimulating nitric oxide synthase expression and the generation of organic peroxides.

Differentiated thyroid cancer and hyperthyroidism are treated with radioiodine. However, when the radioisotope dose exceeds certain limits, the patient must be hospitalized to avoid contact with people that would otherwise be exposed to radiation. It would be desirable to obtain a similar therapeutic effect using lower radioiodine doses. Radiosensitizers can be utilized for this purpose. Nicotinamide (NA) increases thyroid radiosensitivity to 131I in both normal and goitrous glands. NA causes a significant increase in thyroid blood flow, which would increase tissue oxygenation and tissue damage via free radicals. Wistar rats were treated with either nicotinamide (NA), 131I or both. The expression of the three isoforms of nitric oxide synthase (NOS) in the thyroid (Western blot) and the activities of SOD, GPx, catalase and organic peroxides were determined. Treatment with NA or 131I increased the expression of eNOS and the generation of organic peroxides. When administered jointly, they showed a synergistic effect. No changes were observed in the other NOS isoforms or in the activities of catalase, glutathione peroxidase and superoxide dismutase. NA potentiates the effect of 131I by increasing eNOS, which would in turn stimulate NO production, increasing thyroid blood flow and tissue damage via organic peroxides.

Long-term effect of norepinephrine on thyroglobulin gene expression in FRTL-5 cells.

Many types of evidence support a role of the sympathetic nervous system in the regulation of thyroid function, although there is no general consensus on the type of influence that catecholamines exert. Depending on the experimental approach, epinephrine and norepinephrine (NE) can stimulate, inhibit, or fail to act on thyroid function. The aim of this study was to determine the effect of NE on thyroglobulin (Tg) synthesis and gene expression in FRTL-5 cells. Tg content, measured by immunoprecipitation with a specific antibody, showed that NE caused a 45% inhibition of thyrotropin (TSH) effect. The content of Tg mRNA was analyzed by Northern blot, the relative inhibition in total Tg mRNA levels from NE-treated cells, compared to TSH alone, ran parallel with inhibition in Tg content, while total RNA did not change after incubation with NE. There was no alteration in Tg mRNA stability by NE. When plasmids harboring different sequences of Tg promoter fused to the CAT reporter gene were transfected into FRTL-5 cells, TSH treatment stimulated promoter activity while NE diminished this effect by 43%-55%. Northern blots were performed to analyze mRNA for thyroid transcription factors (TTF1, TTF2, Pax8), and no significant changes were observed with the different treatments. In conclusion these results suggest that NE inhibits Tg synthesis at the transcriptional level.

Boron neutron capture therapy for undifferentiated thyroid carcinoma: preliminary results with the combined use of BPA and BOPP.

We have shown the selective uptake of borophenylalanine (BPA) by undifferentiated human thyroid cancer (UTC) ARO cells both in vitro and in vivo. Moreover, a 50% histologic cure of mice bearing the tumor was observed when the complete boron neutron capture therapy was applied. More recently we have analyzed the biodistribution of BOPP (tetrakis-carborane carboxylate ester of 2,4-bis-(alpha,beta-dihydroxyethyl)-deutero-porphyrin IX) and showed that when BOPP was injected 5 days before BPA, and the animals were sacrificed 60 min after the i.p. injection of BPA, a significant increase in boron uptake by the tumor was found (38-45 ppm with both compounds vs. 20 ppm with BPA alone). Five days post the i.p BOPP injection and 1h after BPA the ratios were: tumor/blood 3.75; tumor/distal skin 2. Other important ratios were tumor/thyroid 6.65 and tumor/lung 3.8. The present studies were performed in mice transplanted with ARO cells and injected with BOPP and BPA. Only in mice treated with the neutron beam and injected with the boronated compounds we observed a 100% control of tumor growth. Two groups of mice received different total absorbed doses: 3.00 and 6.01 Gy, but no further improvement in the outcome was found compared to the previous results using BPA alone (4.3 Gy).

Biodistribution of p-borophenylalanine (BPA) in dogs with spontaneous undifferentiated thyroid carcinoma (UTC).

Human undifferentiated thyroid carcinoma (UTC) is a very aggressive tumor which lacks an adequate treatment. The UTC human cell line ARO has a selective uptake of BPA in vitro and after transplanting into nude mice. Applications of boron neutron capture therapy (BNCT) to mice showed a 100% control of growth and a 50% histological cure of tumors with an initial volume of 50 mm(3) or less. As a further step towards the potential application in humans we have performed the present studies. Four dogs with diagnosis of spontaneous UTC were studied. A BPA-fructose solution was infused during 60 min and dogs were submitted to thyroidectomy. Samples of blood and from different areas of the tumors (and in one dog from normal thyroid) were obtained and the boron was determined by ICP-OES. Selective BPA uptake by the tumor was found in all animals, the tumor/blood ratios ranged between 2.02 and 3.76, while the tumor/normal thyroid ratio was 6.78. Individual samples had tumor/blood ratios between 8.36 and 0.33. These ratios were related to the two histological patterns observed: homogeneous and heterogeneous tumors. We confirm the selective uptake of BPA by spontaneous UTC in dogs and plan to apply BNCT in the future.

Comparison of the effects of 3,5,3'-triiodothyroacetic Acid and triiodothyronine on goiter prevention and involution and on hepatic and skeletal parameters in rats.

3,5,3'-triiodothyroacetic acid (TRIAC) has been used to suppress pituitary TSH secretion with reported attenuation of extrapituitary effects. We investigated whether equivalent doses of T (3) and TRIAC preventing the induction of goiter by methimazole (MMI) had a different or similar impact on peripheral tissues, such as liver and bone. In particular, we compared the effects of both compounds on the activity of the hepatic thyroid hormone-responsive enzymes, malic enzyme and L-glicerol-3-P dehydrogenase; bone mineral density and biochemical parameters of bone turnover, such as bone alkaline phosphatase (b-ALP) and the carboxy-terminal telopeptide region of type I collagen (beta-CTX); and the activity of thyroid ornithine decarboxylase (ODC). We also compared the effects of T (3) and TRIAC on the involution of MMI-induced goiter. Our results showed that TRIAC was more effective than T (3) to reduce MMI-induced goiter in a short-term goiter involution assay. TRIAC increased hepatic enzymes activity and beta-CTX levels, a parameter of bone resorption, more than T (3). However, bone mineral density was not altered by either treatment. Both compounds even reduced ODC activity at doses that were not effective at the pituitary level. These results demonstrate increased TRIAC hepatic and antigoitrogenic activity compared to T (3). TRIAC induces an imbalance in bone remodeling without affecting bone mineral density. Further studies are required to clarify this point.

Comparative efficacy and side effects of the treatment of euthyroid goiter with levo-thyroxine or triiodothyroacetic acid.

Euthyroid goiter is usually treated with TSH-inhibitory doses of levo-T(4) (L-T(4)). Because triiodothyroacetic acid (TRIAC) decreases TSH levels, the following study was perfomed: 36 euthyroid goitrous female patients (no cancer or chronic thyroiditis) were randomized to TRIAC (19.6 micro g/kg) (n = 19) or L-T4 (1.7 microg/kg) (n = 17) treatment during 11 months. Goiter volume; lumbar and femoral bone mineral density; serum osteocalcin; deoxypyridinoline; TSH; free T(4); total, high-density lipoprotein, and low-density lipoprotein cholesterol; and triglycerides were measured before and after the study period. Student's t test and chi(2) analysis were performed. TSH values (microunits per milliliter) in the TRIAC and L-T(4) groups were: 1.91 +/- 0.6 (basal) and 0.180 +/- 0.1 (after) and 2.1 +/- 2.5 (basal) and 0.180 +/- 0.3 (after), respectively. Thyroid volume decreased 37.9 +/- 35.4% in the TRIAC patients and 14.5 +/- 39.5% in the L-T(4) group (P = 0.069). Forty-two percent of the goiters with TRIAC reduced more than 50% their initial volume vs. 17.7% with L-T(4) (P = 0.15). With TRIAC, patients experienced fewer side effects. No differences in the changes of bone mineral density, serum deoxypyridinoline, osteocalcin, or the lipid profile were observed between both groups. The present results show that TRIAC is more effective than L-T(4) in the reduction of goiter size, with comparable effects on peripheral parameters.

A new animal model for human undifferentiated thyroid carcinoma.

An animal model of undifferentiated thyroid carcinoma (UTC), which may be useful for studying tumorigenesis and response to new therapies, is described. The UTC human cell line ARO was implanted into the back of the nude mice. The histology, induction of metastasis, and biokinetics of in vivo and in vitro growth, as well as cytogenetic and molecular aspects were studied. The tumor showed extensive viability with high mitotic activity. At 117 days, the tumors reached a size of 1,700 mm(3) and showed a central necrotic portion with a thin layer of viable cells. When the number of passages in the mouse increased the growth rate decreased. The cytogenetic and molecular studies did not show differences between the original line and the sublines that could explain this phenotypic change. Moreover, the original ARO cell line and its sublines showed a complex clonal karyotype including structural alterations with deletions and translocations involving chromosomes 5, 7, 8, 9p, 11p, 17q 19p, and 20q that were consistent with earlier reported data in UTC. This work provides an animal model of UTC pheno- and genotypically similar to the original human tumor, which may be useful for exploring new therapeutic modalities.

Characterization of sex hormone-binding globulin isoforms in hypothyroid women.

Liver sex hormone-binding globulin (SHBG) biosynthesis is regulated by triiodothyronine (T3). This regulation is responsible for increased serum SHBG concentrations in hyperthyroid states. However, in hypothyroidism, normal SHBG levels are frequently found. To understand this we have characterized circulating SHBG isoforms according to their sialic acid content, which determines its half-life, in euthyroid and hypothyroid women. Six euthyroid (aged 56 +/- 8 years) and five hypothyroid women (51 +/- 13 years) were studied. Their body mass index (BMI) range was 20-25. Hypothyroidism diagnosis was based on clinical findings, elevated basal thyrotropin (TSH) and decreased T3 and thyroxine (T4) values. Total SHBG was measured by radioimmunoassay (RIA) and SHBG isoforms were isolated using preparative isoelectrofocusing. For comparisons, two-tailed t test was applied. No statistical difference was found between the total SHBG levels of hypothyroid and euthyroid postmenopausal women. Three groups of SHBG isoforms were isolated in the euthyroid group: S(I): pl: 5.0-5.2: 20% +/- 4%, S(II) : pl 5.2-5.4: 50% +/- 3% and S(III): pl 5.4-5.6: 29% +/- 4%. In hypothyroid patients, although the three groups of isoforms were isolated in the same pH range, S(I) and S(II) proportions were different (p < 0.001) when compared to normal women: S(I): 34% +/- 4%, S(II): 33% +/- 9.9% and S(III): 29% +/- 5.7%. These results show that hypothyroid patients have a higher proportion of more acidic SHBG isoforms. This variation may explain the normal levels of serum SHBG observed in hypothyroidism.

Selective uptake of p-borophenylalanine by undifferentiated thyroid carcinoma for boron neutron capture therapy.

Undifferentiated thyroid carcinoma (UTC) lacks an effective treatment. Boron neutron capture therapy (BNCT) is based on the selective uptake of 10B-boronated compounds by some tumors, followed by irradiation with an appropriate neutron beam. The radioactive boron originated (11B) decays releasing 7Li, gamma rays and alpha particles, and these latter will destroy the tumor. In order to explore the possibility of applying BNCT to UTC we have studied the biodistribution of BPA. In in vitro studies, the uptake of p-10borophenylalanine (BPA) by the UTC cell line ARO, primary cultures of normal bovine thyroid cells (BT), and human follicular adenoma (FA) thyroid was studied. No difference in BPA uptake was observed between proliferating and quiescent ARO cells. The uptake by quiescent ARO, BT, and FA showed that the ARO/BT and ARO/FA ratios were 4 and 5, respectively (p < 0.001). In in vivo studies, ARO cells were transplanted into the scapular region of NIH nude mice, and after 2 weeks BPA (350 or 600 mg/kg body weight) was injected intraperitoneally. The animals were sacrificed between 30 and 150 minutes after the injection. With 350 mg, tumor uptake was highest after 60 minutes and the tumor/normal thyroid and tumor/blood ratios were 3 and 5, respectively. When 600 mg/kg body weight BPA were administered, after 90 minutes the tumor/blood, tumor/normal thyroid, and tumor/distal skin ratios for 10B concentrations per gram of tissue were approximately 3, showing a selective uptake by the tumor. The present experimental results open the possibility of applying BNCT for the treatment of UTC.

The protein kinase C pathway inhibits iodide uptake by calf thyroid cells via sodium potassium-adenosine triphosphatase.

The effect of the phorbol esther phorbol myristate acetate (PMA) on iodide uptake was studied in primary cultures of calf thyroid cells. PMA caused a dose- and time-dependent inhibition of thyrotropin (TSH), forskolin, and db-cAMP stimulation, indicating an effect distal to both TSH receptor and cAMP generation. No action was found on iodide efflux, indicating a selective inhibition of iodide uptake. This inhibition was observed even after 5 minutes of incubation, thus excluding a possible genomic action. Bisindolmaleimide (BS), a specific inhibitor of the protein kinase C (PKC) pathway, reverted the effect of PMA. A similar degree of inhibition of the Na+/K+ adenosine triphosphatase (ATPase) and iodide uptake by PMA was found, thus suggesting a link between both parameters. These results indicate that the PKC pathway inhibits thyroid iodide uptake by an action distal to cAMP generation and probably because of a decrease in Na+/K+-ATPase activity.

Changes in thyroid function in puppies fed a high iodine commercial diet.

Abnormally low(131)I uptakes were noticed in dogs fed with commercial diets at the University Animal Clinic in Buenos Aires, but the total iodine content of eight different commercial diets examined was found to provide an iodine intake above daily requirements. To investigate this anomaly, 18 dogs were distributed into three groups, fed either: (1) a home-prepared diet; (2) a commercial diet; (3) a home-prepared diet supplemented with potassium iodide equivalent to that found in the commercial diet. The(131)I uptake in dogs of groups B and C was significantly decreased, as was basal serum thyroxine (T(4)) and free thyroxine (FT(4)), whereas urinary iodide excretion and serum thyroid stimulating hormone (TSH), were increased. The thyroid releasing hormone (TRH)-TSH test showed an increased response in dogs from group B, while the TRH-T(4)test was inhibited in both groups B and C. The results demonstrate that the excessive amount of iodine present in some commercial diets in Argentina causes a significant impairment of thyroid function and hypothyroidism.

Commercial diet induced hypothyroidism due to high iodine. A histological and radiological analysis.

A number of puppies of the School Hospital of the Faculty of Veterinary Science-UBA showed bone changes. Measurement of the iodine content of the commercial diet showed a significant increase in its content. Iodine excess causes alterations in thyroid function and morphology, and its hormones have a direct action on bone formation. Three groups of puppies were fed on different diets: a home-prepared diet, a commercial diet (containing 5.6 mg potassium iodide/kg dry food), and a home-prepared diet supplemented with 5.6 mg potassium iodide/kg dry food. Groups B and C developed hypothyroidism. A significant decrease (p<0.05) in the styloid apophyseal surface was found in groups B and C vs. A, determined by radiography. Histologically, the hypertrophied cartilage was shorter in groups B and C than in group A (p<0.0001). The present results suggest that commercial diets with a high iodine content may cause hypothyroidism and changes in bone metabolism.

High-affinity binding of T3 to epididymis nuclei.

Thyroid hormones play an important role in epididymal function. Hypothyroid animals experience a significant decrease in the number and forward motility of sperm and a remarkable impairment of epididymal morphology. However, it is yet unknown if such activity is due to direct actions of iodothyronines on the target epididymis. The eventual identification of T3 receptors in the nucleous of epididymal cells becomes relevant. For this reason, the authors searched for specific high-affinity binding of T3 to these nuclei. Twenty prepuberal male Wistar rats were used. The testes and epididymis were approached as one unit through a scrotal incision. The fat-free epididymides were subjected to standard techniques to prepare the nuclei for incubations with 125I-T3 concentrations, ranging from 0.5 x 10(-9) to 2.0 x 10(-11) M. Calculations of association constants and binding capacities were performed according to Scatchard. A single binding site with a Ka of 3.06 +/- 0.6 x 10(9) M(-1) or Kd of 3.26 +/- 0.6 x 10(10) M and a maximal binding capacity of 0.11 +/- 0.02 pmol T3/microg DNA were observed. It is concluded that these nuclei contain a specific T3 receptor. This finding strongly suggests that thyroid hormones have direct effects on the epididymis.

Effect of the interaction of TSH and insulin on the stimulation of 2-deoxyglucose uptake in FRTL-5 cells.

Since thyroid glycogen stores are low, the uptake of glucose is very important in order to maintain cell function (house-keeping). Previous studies have shown that TSH and insulin, independently, are regulators of this parameter. Since their corresponding mechanisms of action are different, we investigated the possible effect of the interaction between TSH and insulin on the stimulation of 2-deoxyglucose (2-DOG) uptake, a non metabolizable derivative of glucose. Confluent FRTL-5 cells were submitted to different treatments, usually for 72 h. In one series of experiments the concentration of TSH was kept constant, at 1 U/l, and the addition of insulin, from 0.16 to 1.6 micromol/l caused a progressive synergic increase in DOG uptake. When insulin concentration was kept constant, increasing amounts of TSH, from 0.5 to 10 U/l), also caused a synergic stimulation of DOG uptake. The effect of insulin was mimicked by IGF-1 (1-10 nmol/l), while that of TSH was mimicked by forskolin. Timecourse studies showed that TSH had a peak at 3 h of incubation, while insulin caused a progressive increase for up to 72 h. At short incubation times, up to 6 h, an additive effect of TSH and insulin was observed, while at longer times the interaction was synergic. The present results suggest that the interaction between the cAMP and the tyrosine kinase pathways on DOG uptake would involve two different mechanisms. At early times the effects of both hormones are additive, while in longer periods it becomes synergic.

Presence of a soluble inhibitor of thyroid iodination in primary cultures of thyroid cells.

Monolayer cultures of thyroid cells lose their iodide organification capacity a few days before the disappearance of thyroid peroxidase (TPO) activity. The present studies were performed in order to clarify this point. The above mentioned difference was due to the presence of an inhibitor in the monolayer thyroid cells culture, given that total homogenate prepared from confluent cells caused a significant inhibition of activity of TPO from fresh tissue. The inhibitor was localized in the 105000g supernatant of the homogenate of the cell culture, but not in a similar preparation obtained from fresh thyroid. It is thermostable, dialyzable and has a molecular weight of less than 2 kDa. Addition of the inhibitor at the end of the reaction of tyrosine iodination failed to alter the results. This fact suggests that the compound does not destroy the iodinated product. The presence of the cytosolic inhibitor was observed in monolayer thyroid cell cultures of different species (bovine, porcine, rat and human) but not in free follicles cultures.

Partial characterization of guanylyl cyclase activity in calf thyroid.

The aim of the present study was to perform the partial characterization of the enzyme guanylyl cyclase (GC) in bovine thyroid. The results obtained showed the presence of two types of GC: one is soluble and comprises around 79% of total activity, while the other is particulate. Treatment with 1% Triton X-100 increased both activities. When the kinetics of the enzyme was analyzed, using the complex MnGTP as a substrate, the results showed a Michaelis type kinetics for the soluble enzyme, with a Km of 0.037 mM, whereas the particulate GC showed a positive allosteric behavior with a S0.5 of 0.214 mM and a Hill coefficient of 1.9, indicating that the enzyme has at least two binding sites for the substrate. When the influence of different Mn2+ concentrations was studied, a positive allosteric behavior for the soluble GC was found, with a S0.5 of 1.2 mM and a Hill coefficient of 2.2. The kinetics of the particulate enzyme under similar conditions was of Michaelis type, with a Km of 0.752 mM. Although the enzyme is highly dependent on Mn2+, it was of interest to investigate the possible effects of other divalent cations, such as Ca2+ and Mg2+. The replacement of Mn2+ for Mg2+ caused a complete disappearance of the particulate enzyme activity, while the soluble activity decreased by 85%. Addition of Ca2+ had no effect on either GC. However, with suboptimum. concentrations of Mn2+, high Ca2+ concentration caused an increase in soluble activity, but it comprised only 20% of maximum activity with optimum Mn2+ concentrations. With the particulate enzyme a slight but significant inhibition was observed.

Variations of sex hormone-binding globulin in thyroid dysfunction.

With the aim of understanding the variations of the levels of sex hormone-binding globulin (SHBG) in thyroid dysfunction, we studied the influence of factors that also modify SHBG, such as menopausal status, age, and body mass index (BMI) in women with hypothyroidism and hyperthyroidism, both overt and subclinical. Statistical analysis was performed by means of analysis of variance (ANOVA), stepwise multiple regression, and partial correlation. The ANOVA showed a significant statistical difference among the means of SHBG of all groups (p<0.01). The difference was due to the group that included hyperthyroid women. Multiple regression analysis showed that the main factors influencing SHBG were BMI and age, except for the hyperthyroid group, where the most important independent variables were triiodothyronine (T3) and thyroxine (T4). Partial correlation controlling the effect of BMI and age showed no association between SHBG and the other variables in all groups except for the subclinical hyperthyroid and hyperthyroid, where we found a significant association between SHBG and T4 and T3. The premenopausal or postmenopausal status did not modify SHBG levels. When the patients are taken as a whole, BMI, age, T4, and T3 all have an association with SHBG levels according to the multiple regression analysis.