Sensitive assay to detect thyroid stimulating antibody (TSAb) in the presence of thyroid stimulation blocking antibody (TSBAb) in serum.

The detection of thyroid stimulating antibody (TSAb) activity in the presence of thyroid stimulation blocking antibody (TSBAb) in Graves' serum is difficult because TSBAb blocks TSAb activity. We recently demonstrated that polyethylene glycol (PEG) augments TSAb activity in porcine thyroid cells (PTC) assay. This PEG-induced augmentation makes it possible to develop a sensitive assay to detect TSAb in the presence of TSBAb. We studied the effects of PEG on TSAb- and TSBAb-activities in PTC using 4 different preparations of the samples; (1) crude IgG using PEG 22.5% precipitated fraction (PF) from Graves' serum (0.2 ml), (2) crude IgG using PEG 12.5% PF, (3) serum (50 microl), and (4) serum (50 microl) in the presence of 5% PEG (final). When the effects of PEG on TSAb activity using crude IgG were examined, PEG 22.5% PF showed significantly higher TSAb activity than PEG 12.5% PF as reported previously. The augmentative effect of PEG on TSAb activity was also observed by the addition of 5% PEG to serum. We also demonstrated that PEG augmented TSAb-activities even in TSBAb-positive serum by two methods (crude IgG using PEG 22.5% PF and the addition of 5% PEG to serum). TSBAb activities were expressed by two calculation methods (A= [1 - (a - b)/(c - d) x 100] and B = [1 - (a - d)/(c - d) x 100], where a is cAMP produced in the presence of bTSH and patient's IgG, b is cAMP produced in the presence of patient's IgG, c is cAMP produced in the presence of bTSH and normal IgG, and d is cAMP produced in the presence of normal IgG). In the presence of TSAb, the values of A method were always higher than those of B method, since TSAb stimulated cAMP synthesis. We have developed two sensitive methods to detect TSAb even in the presence of TSBAb in serum using PEG; 1) incubation of crude IgG using PEG 22.5% PF from serum (0.2 ml), and 2) co-incubation of 5 % PEG with test serum (50 microl).     

Regulation of mitochondrial protein turnover by thyroid hormone(s)

1. The effect of thyroidectomy on turnover rates of liver, kidney and brain mitochondrial proteins was examined. 2. In the euthyroid state, liver and kidney mitochondria show a synchronous turnover with all protein components showing more or less identical half-lives compared with the whole mitochondria. The brain mitochondrial proteins show asynchronous turnover, the soluble proteins having shorter half-lives. 3. Mitochondrial DNA (m-DNA) of liver and kidney has half-lives comparable with that of whole mitochondria from these tissues. 4. Thyroidectomy results in increased half-lives of liver and kidney mitochondria, with no apparent change in the half-life of brain mitochondria. 5. A detailed investigation of the turnover rates of several protein components revealed a significant decrease in the turnover rates of mitochondrial insoluble proteins from the three tissues under study. 6. The turnover rates of m-DNA of liver and kidney show a parallel decrease. 7. Thus it is apparent that thyroid hormone(s) may have a regulatory role in maintaining the synchrony of turnover of liver and kidney mitochondria in the euthyroid state. Turnover of brain mitochondria may perhaps be regulated by some other factor(s) in addition to thyroid hormone(s). 8. It seems likely that during mitochondrial turnover m-DNA and insoluble proteins may constitute a major unit. 9. The mitochondrial protein contents of the three tissues are not affected by thyroidectomy. 10. No correlation was seen between the turnover rate of mitochondria and cathepsin activity in any of the tissues under study in normal or thyroidectomized animals. 11. On the other hand, mitochondrial proteinase activity shows good correlation with the turnover rates of mitochondria in normal animals, and a parallel decrease in activity comparable with the decreased rates of turnover is observed after thyroidectomy. 12. It is concluded that mitochondrial proteinase activity may play a significant role in their protein turnover.     

Inhibition of TSH binding to chicken thyroid by some cases of LATS (long acting thyroid stimulator)

TSH receptor antibody (TRAb) activity using chicken thyroid receptor (c-TRAb) and porcine thyroid receptor (p-TRAb) was determined by the incubation of 125I-bovine TSH with each receptor. Both c-TRAb and p-TRAb activity in LATS positive and negative Graves' sera were compared. 15 out of 39 LATS positive sera and 4 out of 46 LATS negative sera had positive c-TRAb activity. On the other hand, all LATS positive sera and 33 out of 46 LATS negative sera had positive p-TRAb activity. No relationship between c-TRAb and p-TRAb activity was observed, and there was also no correlation between c-TRAb and LATS activity. Changes in c-TRAb, p-TRAb and LATS activity in the clinical course of patients with Graves' disease were examined. These activities were parallel in some cases, but in others they were not. A weak c-TRAb activity was observed in 4 out of 29 Hashimoto's disease, but all cases with thyroid cancer and subacute thyroiditis showed no activity. Sera with positive c-TRAb activity did not stimulate chicken thyroid in chick bioassay. These results suggest that some cases of TRAb in Graves' disease (mainly LATS) inhibit TSH binding to chicken thyroid receptor (non-mammalian species) in the same way as mammalian thyroid, but may not have any stimulatory action on thyroid hormone synthesis. It is interesting to note that TRAb including LATS have the similar effect on TSH receptor even in nonmammalian species.     

Measurement of thyroid stimulating hormone (TSH) in human urine

Using a highly sensitive and specific immunoradiometric assay kit for human TSH, we measured TSH concentrations in unprocessed urines in normal subjects, in patients with primary hypothyroidism, and patients with renal disease. In five of ten normal subjects TSH was detectable in urine samples (less than 20-69 microU/day). In five patients with hypothyroidism, the urinary TSH excretion was increased. In seven out of ten patients with nephrotic syndrome, eight out of nine patients with chronic renal failure and two patients with tubular dysfunction, the urinary TSH excretion was increased. The urinary TSH excretion correlated significantly with both urinary protein excretion and urinary beta 2-microglobulin excretion. These results suggest that the renal handling of TSH involves both glomerular filtration and tubular re-absorption, and that urinary TSH excretion is increased when serum TSH is increased and either glomerular or tubular function is impaired.     

Localization of transferrin receptors (TFRs) in human non functioning thyroid nodules and in extranodular thyroid tissue

In this research the TFR localization in non functioning human thyroid nodules and in the extranodular thyroid tissue, using an immunohistochemical technique, has been studied. For this study a monoclonal antibody (B3/25) against TFR and the peroxidase technique have been utilized. Moreover a morphometric comparative analysis was carried out based on the following parameters: 1) mean immunoreactive area for microscopic field, 2) mean value of immunoreactive follicular perimeter, 3) integrated optical density, 4) % of immunoreactive area on total examined area in nodular and extranodular tissue. The immunoreactivity was detected in some follicular cells in a number of follicles randomly distributed in the extra nodular tissue. As concern the non functioning thyroid nodules, the positivity was localized in the generality of the follicles both in the flattened epithelial cells of the larger follicles and in the cuboidal cells of the smaller ones. The morphometric parameters confirm a statistically significant difference of immunoreactivity between extranodular and nodular tissue. These results suggest that TF might play a role in the cellular proliferation of thyroid gland.     

Circulating concentrations of thyroid hormones in pregnant camels (Camelus dromedarius )

Blood samples from 16 female camels were collected at monthly intervals commencing from 60 d post. breeding until the last month of gestation. Two camels failed to conceive and two had unnoticed abortions. The average gestation period was 398+/-13 and 372+/-11 in camels bearing male and female fetus, respectively, with an overall mean of 383+/-9 d. Sera were analyzed for thyroxine (T(4)) and triiodothyronine (T(3)) by radioimmunoassay. Mean T(4) and T(3) concentrations varied from 76 to 116 ng/ml and 0.73 to 1.32 ng/ml, respectively, during various stages of gestation. In general, the T(4) and T(3) levels were higher during early pregnancy, with lowest values in the tenth month. T(4):T(3) ratio showed minor, nonsignificant fluctuations. Age of dam of sex of fetus had no effect on hormone levels. Similarly, hormone levels were not affected by failure of conception or by abortion.     

Regulation of mitochondrial membrane protein turnover by thyroid hormone(s)

Effects of thyroidectomy on turnover rate of proteins of rat liver mitochondria, mitochondrial membranes and microsomes were examined. Thyroidectomy resulted in a significant increase in the half-lives of whole mitochondria and inner membrane-matrix, the effect being less pronounced on the half-lives of outer mitochondrial membrane and microsomes.