We examined a method for the measurement of total, activated and non-activated glucocorticoid receptors using sodium-p-hydroxymercuribenzoate (PHMB) and dithiothereitol (DTT) developed by Banerji and Kalimi (1981).
Since the concentration of PHMB required for dissociation of the ligand from the receptors varied with the concentration of protein in the reaction mixture and the rate of reassociation of the ligand to the ligand-liberated receptors was sensitive to the concentration of PHMB used, it was necessary to find the minimum concentration of PHMB which was required for complete dissociation of the ligand. When the optimum concentration of PHMB was selected based on the concentration of protein in the cytosol, almost 100% exchange was attained in the non-heated dexamethasone (Dex)-receptor complexes by this method. However when Dex-receptor complexes were heated at 25° for 30min, the amount of 3H-Dex reassociated with the glucocorticoid receptors dropped to 60% of that of the non-heated ones. DEAEcellulose chromatography of the heated sample revealed that approx. 40% of the bound receptors were activated (eluted with 0.05 M KCl) during the heating period. After DEAE cellulose column chromatography of the exchanged ³HDex receptor, complexes reassociated with ³H-Dex were observed only in the fraction of unactivated receptor complexes (eluted with 0.2 M KCl). Furthermore, the fraction eluted with 0.05M KCl in the DEAE cellulose chromatography of liver cytosol bound to unlabelled Dex did not exchange significantly with ³H-Dex with the method used in the present study. These results all consistently indicated that in the activated receptor complexes dissociation of the ligand was performed almost completely, but reassociation was not achieved successfully after treatment with PHMB and DTT.
From these observations, the method described by Banerji and Kalimi does not seem to be suitable in assessing the bound-activated receptor complexes in tissues.

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A penicillinase linked enzyme immunoassay was developed for the estimation of pregnanediol-3 α-glucuronide (PdG) in urine. The immunoassay satisfied all the validity criteria and was used in detecting ovulation and in the assessment of corpus luteal function (CLF) during spontaneous or induced cycles. Reference values were established by estimating PdG levels in daily early morning urine samples during 31 menstrual cycles obtained from 17 regularly menstruating women. A PdG value of 1.7μg/mg creatinine (μg/mgC)(90th Centile of follicular phase) in any MLP (mid-luteal phase) sample was considered as indicating ovulation. A value of 4.6μg/mgC (20th centile of MLP) was considered to be evidence of sufficient CLF. When this approach was applied to 20 infertile cases, detection of the occurrence of ovulation/anovulation was made correctly in 19 out of 20 cases (95%). Accuracy was poor (55.6%) when the aim of the diagnosis was corpus luteal deficiency. Higher accuracy (88.9%) for corpus luteal deficiency/corpus luteal adequacy was obtained when the sum of PdG concentrations in three MLP samples were taken into consideration. A total of 13.8μg/mgC (thrice the 20th centile for MLP) indicated probable corpus luteal deficiency, and values above this limit were considered to indicate corpus luteal adequacy.

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The serum thyroglobulin (Tg) concentration was measured in 97 patients with diabetes mellitus (39 males, 58 females). Hyper Tg-nemia which exceeds the normal range (1.0-26.6ng/ml) was observed in 10 patients (3 out of 21 cases treated with diet alone, 3 out of 50 cases treated with oral hypoglycemic agents, 4 out of 26 cases treated with insulin). There was no significant correlation between concentrations of serum Tg and triiodothyronine (T₃), thyroxine (T₄), fasting plasma glucose (FPG), and hemoglobin A_1c (HbA_1c). However, a positive correlation was observed between serum concentrations of Tg and thyroid stimulating hormone (TSH). Patients with diabetes were divided into three groups according to the mode of treatment (Group I; diet alone, n=21, Group II; oral hypoglycemic agents, n=50, Group III; insulin, n=26). No significant difference in the serum Tg concentration was observed among the three groups. They were also divided into two groups; normal Tg-nemia (Group A, n=87) and hyper Tg-nemia (Group B, n=10). There was no difference between levels of T₃, T₄, FPG, and HbAic in the two groups. The serum TSH concentration measured by double antibody RIA and two site immunoradiometric assay in Group B was significantly higher than that in Group A. These results suggest that hyper Tg-nemia in patients with diabetes could be due to the increased TSH concentration which reflects latent subclinical primary hypothyroidism in them.

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The effect of NADPH and Ca²⁺ on 3, 5, 3'-L-triiodothyronine (T₃) binding to cytosolic T₃ binding protein (CTBP) in rat kidney was investigated in vitro. Extraction of rat kidney cytosol with 10% charcoal at 4° for 30min. inactivated specific T₃ binding. The decreased T₃ binding activity in extracted cytosol could be restored by adding NADPH or Ca²⁺ to the incubation medium. Each substance increased the capacity for T₃ without changes in the affinity for T₃. The T₃ binding was maximally increased by 25μM NADPH or by 1.0mM Ca²⁺ in the presence of 0.1mM EDTA. The increase in T₃ binding, induced by 25μM NADPH, was enhanced by adding 0.2-1.0mM Ca²⁺ in a concentration dependent-manner. The increase in T₃ binding, induced by 1.0mM Ca²⁺, was also enhanced by adding 3.125-25.0μM NADPH. The NADPH-induced increase in T₃ binding capacity was amplified by Ca²⁺ in a multiplicative manner. The results suggested that Ca²⁺ cooperatively augmented an NADPH function in cytosolic T₃ binding.

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To clarify whether hyperinsulinemia accelerates sympathetic nervous system (SNS) activity, norepinephrine (NE) turnover, a reliable indicator of SNS activity, was measured in the interscapular brown adipose tissue (IBAT) and heart of hyperinsulinemic yellow KK and normoinsulinemic C57BL control mice at 12 weeks of age. The yellow KK mice were more obese and had higher levels of plasma glucose (about 2.3 times) and of plasma insulin (about 24 times) than did the control mice. In IBAT, the rate of NE turnover following blockade of NE synthesis with α-methyl-p-tyrosine (α-MPT) was significantly slower in yellow KK mice than in C57BL mice, although in heart, no significant difference between both groups was observed in NE turnover. These results suggest that hyperinsulinemia dose not always increase NE turnover, and furthermore that the reduced NE turnover in IBAT of yellow KK mice may be one of the important factors in the development of obesity of this animal, as it is recognized that brown adipose tissue is a main effector of diet-induced thermogenesis and its defect or absence would predispose to obesity.

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A case of systemic lupus erythematosus (SLE) associated with Sjögren's syndrome had extremely low serum triiodothyronine (T₃) with normal levels of serum thyroxine (T₄) measured by single antibody radioimmunoassays (RIAs) and thyroid stimulating hormone (TSH) during steroid treatment. Measurement of serum T₃ and T₄ with double antibody RIAs showed unusually high T₃ and normal T₄ concentrations. Examination of her serum revealed the presence of IgG class anti-T₃ autoantibodies whose Scatchard plot was analyzed in two components; one with a higher associate constant (8.6×10⁸M^-1) and a lower binding capacity (5.6×10^-7mol/ml serum); the other a lower associate constant (3.5×10⁷M^-1) and a higher binding capacity (2.1×10^-6mol/ml serum). Antithyroglobulin (Tg) autoantibody has been positive throughout the seven year observation period. A significant positive correlation between titers of anti-Tg autoantibodies indicated that the antigen of anti-T₃ antibodies in the patient could be T₃ containing antigenic site (s) on the Tg molecule.

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Calmodulin (CaM) antagonists, W-7 and W-5, chlorpromazine and haloperidol, especially W-7 inhibited ¹²⁵I-bovine TSH binding to human and porcine thyroid receptors dose-dependently in the presence of calcium ion. This inhibitory effect of W-7 was diminished by the addition of ethylenglycol tetraacetic acid (EGTA) or ethylendiamine tetraacetic acid (EDTA).
CaM also dose-dependently inhibited the binding of ₁₂₅I-TSH to thyroid receptor in the presence of calcium ion. TSH binding to thyroid receptor was completely inhibited by more than 30μg of CaM, and this inhibition was abolished by adding EGTA. On the other hand, the antibody to CaM inhibited partially ¹²⁵I-TSH binding to its receptor.
These results suggest involvement of CaM and CaM-binding sites in the TSH receptor of thyroid.

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We detected an antibody to calmodulin (CaM) in sera from patients with Graves' disease. Four sera out of 300 from patients with Graves' disease demonstrated increased CaM binding activity as compared with 300 sera from normal subjects, while no binding activity was detected in sera from autoimmune thyroiditis. The binding could be demonstrated as due to the antibody to CaM by the double antibody method, polyethyleneglycol method, gel filtration and radioimmunoelectrophoresis, respectively. These antibodies were thought to be polyclonal immunoglobulins (IgG and/or IgA).
CaM has proven to be a poor antigen because of the structural identity of CaM from different species. The incidence of the antibody to CaM in Graves' disease is low and the pathophysiological significance of this antibody to CaM had remains obscure.

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It is still unclear whether, in the male rat, castration and androgen replacement affect the binding characteristics of brain opiate receptors. To clarify this issue, the effects exerted by orchidectomy and testosterone (T) replacement on the subpopulation of brain μ opiate receptors were studied in male rats; testosterone was administered via subcutaneous Silastic capsules. Utilizing ³H-dihydromorphine (a μ receptor ligand) it has been shown that the affinity constant (Ka) of brain μ opiate binding sites, measured in plasma membrane preparations, is not affected by castration. When μ receptor concentrations were measured in individual brains, it was found that gonadectomy and T replacement failed to produce any change in the number of μ opiate receptors. These data suggest that, in male rats, gonadal steroids do not develop their central feedback effects by affecting brain μ opiate receptors.

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Ten patients with liver cirrhosis and six normal subjects were studied to evaluate the effect of iopanoic acid (IA) on thyrotropin secretion. A thyrotropin-releasing-hormone (TRH) test was performed before and 5 days after IA administration (single oral dose of 3g). After IA administration, a significant increase in TSH response to TRH was observed in normal subjects. In cirrhotics, however, it did not significantly increase after IA administration. The serum T3 and T3/TBG ratio were significantly decreased and the serum T4 and T4/TBG ratio were increased after IA administration in normal subjects and cirrhotics. There was no significant difference in the % decrease in serum T3, % increase in serum T4 or other thyroid hormone parameters including TSH in IA induced TSH responders (R) and non-responders (NR). However, r-T3 before and after IA in R was higher than those in NR. The values for hepatic function tests such as serum albumin, prothrombin time, 45 minutes retention rate of bromsulphalein (BSP_{45 min}) and the cholinesterase (ChE) level in R were not different from those of NR.
These results suggested that in cirrhotics, abnormal regulation of the hypothalamo-pituitary system might exist.

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Galactorrhea was found in 5 patients with subclinical hypothyroidism. The galactorrhea consisted of the discharge of a few drops of milk only under pressure. Serum T₄ was in the lower level of the normal range, but serum T3 was normal (T₄: 6.3±1.2μg/dl, T₃: 113±7ng/dl). Basal serum TSH and PRL were slightly increased only in 2 and 1 cases, respectively. The PRL responses to TRH stimulation were exaggerated in all cases, although the basal levels were normal. An enlarged pituitary gland was observed in 1 patient by means of CT scanning.
All patients were treated by T₄ replacement. In serial TRH tests during the T₄ replacement therapy, the PRL response was still increased even when the TSH response was normalized. Galactorrhea disappeared when the patients were treated with an increased dose of T₄ (150-200μg/day). Recurrence of galactorrhea was not observed even though replacement dose of T₄ was later decreased to 100μg/day in 4 cases.
In patients with galactorrhea of unknown origin, subclinical hypothyroidism should not be ruled out even when their serum T₄, T₃, TSH and PRL are in the normal range. The TRH stimulation test is necessary to detect an exaggerated PRL response, as the cause of the galactorrhea. To differentiate this from pituitary microadenoma, observation of the effects of T₄ replacement therapy on galactorrhea is essential.

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A 58-year-old woman was admitted to our hospital complaining of headache, dizziness and intermittent elevation of blood pressure. Multiple cafe-au-lait spots and neurofibromas had appeared on the back and the limbs since the age of 30 years. At the age of 54 years she underwent total thyroidectomy because of papillary carcinoma of the thyroid gland. On admission, the levels of plasma norepinephrine and epinephrine, urinary norepinephrine and normetanephrine were all within the normal range. However, urinary excretion of metanephrine was markedly increased to 1.49±0.45 (Mean SD) mg/day and that of epinephrine was also slightly increased. The computed tomographic scans of the abdomen and the scintigraphy with ¹³¹I-metaiodobenzylguanidine revealed a tumor mass in the region of the right adrenal gland. The tumor was histologically confirmed to be pheochromocytoma at the operation. In her family history, her mother and one of her two sisters had von Recklinghausen's disease and another sister suffered from follicular carcinoma of the thyroid gland. As far as we know, this paper is the first report of a patient with von Recklinghausen's disease associated with both pheochromocytoma and non-medullary carcinoma of the thyroid gland, and her family.

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Suppression of TSH and thyroid radioiodine uptake by doses of either T4 or T₃ were compared in 33 patients in whom Graves' thyrotoxicosis had been treated with thioamide drugs and the medication was discontinued for at least 4 months. Thyroidal radiodine uptake was suppressed in 19 patients and was not suppressed in the remaining 14 patients. Basal TSH levels before suppression were 2.07μU/ml in the former, significantly exceeding those of the latter (0.91 μU/ml). A TSH level of at least 1.2μU/ml befbre suppression is a good predictor of positive thyroid radioiodine suppression with a predictive value of 76%. A level lower than 0.7μU/ml before suppression is a good predictor of negative thyroid radioiodine uptake suppression with a predictive value of 89%. The determination of TSH levels before the thyroid suppression test was helpful in predicting the result, but there were limitations.
In the thyroid suppression test positive group, circulating T₄ was depressed by doses of T₃. In them, the magnitude of T₄ depression correlated with the levels of thyroid radioiodine uptake before suppression. The levels of TSH correlated neither to changes in T₄ nor to those in thyroid radioiodine uptake. This indicates that the thyroid glands which show high radioiodine uptake are sensitive to TSH and are also sensitive to suppression. The elevated sensitivity to TSH probably warrants the disappearance of abnormal thyroid stimulation more precisely.

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Serum C-peptide responses to glucagon and daily urine C-peptide excretion in successive periods of different treatment in two groups of patients with non-insulin-dependent diabetes mellitus (NIDDM)(mean interval between two tests<1 month) were compared. In group A patients (n=8), the glycemic control was improved after transferring the treatment from sulfonylurea (SU) to insulin (fasting plasma glucose: SU: 192±47, insulin: 127±21mg/dl, mean±S.D., p<0.01). Fasting serum C-peptide immunoreactivity (CPR) was significantly lower at the period of insulin treatment (SU: 1.93±1.01, insulin: 1.47±0.79ng/ml, p<0.05), but there was no difference in the increase in serum CPR (maximal-fasting)(Δ serum CPR) during glucagon stimulation in the two periods of treatment (SU: 1.70±0.72, insulin: 1.47±0.98ng/ml). In group B patients (n=7), there was no significant difference in glycemic control after transferring the treatment from insulin to SU (fasting plasma glucose: insulin: 127±24, SU: 103±13mg/dl). Fasting serum CPR was significantly lower during the period of insulin treatment (insulin: 1.39±0.64, SU: 2.21±0.86ng/ml, p<0.025), but Δ serum CPR during glucagon stimulation still showed no significant difference between the two periods (insulin: 1.97±1.16, SU: 2.33±1.57 ng/ml). On the other hand, daily urine CPR excretion was constantly lower during insulin treatment than during SU treatment in both groups (group A: SU: 65.6±23.2, insulin: 37.1±15.3μg p<0.01, group B: insulin: 40.3±14.7, SU: 65.6±12.4μg, p<0.01). The data suggest that basal and daily pancreatic B-cell secretion in NIDDM patients varies within the short periods of different treatment. However, the CPR response to glucagon remains stable.

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To assess the clinical value of a sensitive immunoradiometric assay for TSH (IRMA-TSH), serum IRMA-TSH levels were compared with those of a radioimmunoassay (RIA-TSH) in twenty-eight patients with congenital hypothyroidism. Among 144 samples taken from them, 44 samples showed undetectable RIA-TSH, while only 10 samples were undetectable by IRMA-TSH. In two patients prospectively followed, RIA-TSH levels were undetectable when serum T3 and T4 were normal. IRMA-TSH levels, however, were detectable when serum T4 levels were elevated or normal.
The basal RIA- and IRMA-TSH levels in 4 groups (22 patients) were compared and classified according to the TSH response to TRH. The RIATSH levels were undetectable in any in group 1 (n=7; absent response) or group 2 (n=5; low response). At the same time, IRMA-TSH levels were undetectable in only three patients in group 1. In group 3 (n=16; normal response), RIA-TSH levels were undetectable in three, whereas IRMA-TSH levels were detectable in all. The IRMA- and RIA-TSH levels rose in all in group 4 (n=15; exaggerated response).
These results suggest that the serum basal IRMA-TSH levels indicate the responsiveness of TSH to TRH more accurately than basal serum RIA-TSH levels. Therefore, it was concluded that IRMA-TSH may obviate the need for a TRH test and simplify the evaluation of adequate dosage in patients with congenital hypothyroidism during thyroxine treatment.

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Two pituitaries from 7-week-old female rats (Sprague-Dawley strain) were grafted under the capsule of the left kidney of a 49-day old male rat. The pituitary grafted and sham-operated rats were hypophysectomized at 56 days of age. The hypophysectomized rats were given daily injections of NIAMDDoFSH-13 (20μg/0.5ml saline), NIAMDD-oLH-23 (9μg/0.5ml saline) or saline for 4 days starting from day 58. The treated rats and normal male rats were killed at 61 days of age. Testicular homogenates were incubated with [¹⁴C] 4-androstene-3, 17-dione or [³H] progesterone, and enzyme activities per testes were estimated.
Hypophysectomy caused significant decreases in activities of testicular 17β-oxidoreductase and 17-hydroxylase. The decreased activity of 17β-oxidoreductase was significantly stimulated by FSH or LH treatment, whereas the decreased 17-hydroxylase activity was stimulated only by LH treatment. Although pituitary grafts alone showed little or no effect on these enzyme activities in the hypophysectomized rats, the grafts significantly inhibited FSH-stimulated 17β-oxidoreductase activity and the LH-stimulated 17β-oxidoreductase and 17-hydroxylase activities but enhanced LH-induced 5α-reductase activity. The present results confirm previous findings that an excess of prolactin directly inhibits LH-stimulated 17-hydroxylase activity but enhances LH-induced 5α-reductase activity in the rat testis. The present results also demonstrate that the same grafts directly inhibit FSH-stimulated 17β-oxidoreductase activity but have no effect on FSH-induced 5α-reductase activity.

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Hyperthyroidism, microscopic hematuria, and proteinuria developed in an 11-year-old girl. Proteinuria decreased during treatment of hyperthyroidism with an antithyroid drug. On admission, serum anti-thyroglobulin antibody, antimicrosomal antibody, and immune complex were present. The thyrotropin binding inhibitory immunoglobulin (TBII) level was low. On the other hand, an antibody to the ganglioside component (fucosyl-GM1) was detected by an enzyme linked immunosolvent assay (ELISA).
interstitial fibrosis. A renal biopsy specimen showed marked proliferation of A thyroid biopsy specimen showed massive lymphocytic infiltration and mesangial cells and increased mesangial matrix with focal segmental capillary wall abnormality. Electron microscopec studies demonstrated mild paramesangial dense deposits.
A thyroid biopsy specimen showed massive lymphocytic infiltration and interstitial fibrosis. A renal biopsy specimen showed marked proliferation of mesangial cells and increased mesangial matrix with focal segmental capillary wall abnormality. Electron microscopec studies demonstrated mild paramesangialdense deposits.
By indirect immunofluorescence, granular glomerular basement membraneand mesangial staining were not detected with rabbit antibody to thyroglobulin, but were detected with rabbit antibody to fucosyl GM1. Fucosyl GM1 was also seen along the basilar aspect of the thyroid follicular epithelial cells.
These observation suggests the development of glomerulonephritis mediated by thyroid antigen, particularly ganglioside component.

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A reconstructed monolayer was formed using epithelial cells from normal mouse kidney to investigate the hormonal effect on phosphate transport by the renal cells. The cells, when cultured on a Millipore filter, formed a monolayer with an apical negative transepithelial potential of 8.4±0.4mV. When radioactive phosphate was added onto the apical surface of the monolayer (corresponding to the luminal surface of a renal tubule), the phosphate was transported through the cell layer to the basolateral surface (corresponding to the peritubular surface of a renal tubule). This transport process was saturable, energy-dependent, and inhibited by 2, 4-dinitrophenol or ouabain. Dose-dependent parathyroid hormone-induced inhibition (73% of the control) was also evident in this system. Similar inhibition (69% of the control) was observed with DBcAMP. Thus, monolayers reconstructed from cultured mouse kidney cells show characteristics similar to those of renal tubules.

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In order to observe the effect of triiodothyronine (T₃) or thyroxine (T₄) on glucose-induced insulin release from pancreatic islets, isolated pancreatic islets from Wistar male rat were incubated in modified Krebs-Ringer bicarbonate solution (0.2% BSA, pH 7.4) with 3mM, 5mM or 10mM glucose for 60min with or without exposure to T₃ (2.5μg/ml or 7.5μg/ml) or T₄ (2.5μg/ml or 7.5μg/ml). Direct exposure of isolated pancreatic islets to T₃ or T₄ did not have any effect on glucose-induced insulin release from pancreatic islets during 60 min incubation.
But, in successive 60 min incubations following 60 min rest periods after exposure to T₃ or T₄ in the first incubation, glucose-induced insulin release from isolated pancreatic islets previously exposed to T₄ was reduced at 5mM and 10mM glucose concentrations and previous exposure to T₃ had no effect on glucose-induced insulin release. A delayed effect of T₄ on the mechanism of insulin secretion from pancreatic islets was suggested.

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In a primary culture of bovine adrenal zona glomerulosa cells, insulin-like growth factor I (IGF-I)/somatomedin C stimulated DNA synthesis, as measured by [³H] thymidine uptake, at concentrations of 10^-9 and 10^-7M. IGF-I also prevented ACTH-induced suppression of [³H] thymidine uptake. IGF-I in no way affected aldosterone secretion during short-term exposure to cultured cells, however. Our findings suggest that IGF-I plays an important role in the proliferation of adrenal zona glomerulosa cells.

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When monkey hepatocarcinoma cells (NCLP-6E) were treated with 10% of various serum preparations for 24h at 37°C, nonphenolic ring deiodinase activity increased 2.0- to 2.3-fold. Phenolic ring deiodinase activity also increased 0.9- to 2.1-fold. Dialysis of the sera enhanced the effect on deiodinase activities in some preparations, but reduced activity in other serum preparations. Similarly, a 1.3- to 3.1-fold increase in phenolic ring deiodinase activity was observed in rat hepatoma cells (R-Y121B). In this case, dialysis usually reduced the effect of the sera. It is concluded that both large molecule (s)(undialyzable) and small molecule (s)(dialyzable) in serum contribute to the regulation of phenolic and nonphenolic ring deiodinase activity in NCLP-6E and R-Y121B cells.

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A 10-year-old child with idiopathic growth hormone deficiency was treated with recombinant methionyl human growth hormone (m-hGH, Somatonorma^®) at a dose of 14IU/week. Height increased from 122.3 to 126.5cm during the first 9 months of treatment (5.6cm/y), but only from 126.5 to 126.6cm during the next 3 month of treatment (0.4cm/y). Anti-hGH antibody was detected at 2 months of treatment, reached its maximum at the end of 9 months with a titer of 10⁶ and a binding capacity of 2.0mg/L. After switching from m-hGH to pituitary extracted hGH (p-hGH) treatment, his height increased (5.6cm/y). The cause of growth attenuation during m-hGH treatment was again from 126.6 to 132.2cm during the next 12 months of p-hGH treatment concluded to be the high titer of anti-hGH antibody. This is the first case in Japan and one of only three cases in which growth attenuation occurred during m-hGH treatment.

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