The CIBA is very useful in quantifying hormone secretion from single endocrine cells. Variations of this assay can extend its power for evaluating the proportion of hormone-secreting cells in a cell population, determining the responsiveness of individual cells to a particular secretagogue and identifying multihormones that are simultaneously secreted by the same cells. By using the CIBA and its variations, mammotrophs were shown to be heterogeneous with respect to the responsiveness to DA and TRH and cosecretion of PRL and GH. Furthermore, the fact that SP secretion from pituitary cells was increased by thyroidectomy via an increase in the number of SP-secreting cells indicates the possibility that the CIBA can clarify mechanisms underlying changes in hormone secretion and intercellular signaling at the cellular basis.

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A 27-yr-old woman was referred for evaluation of acromegaly and hyperprolactinemia. She had undergone left adrenalectomy at 12 and right adrenalectomy at 17 for Cushing's syndrome due to adrenocortical nodular hyperplasia. At this time a pituitary tumor was found by brain computerized tomography, but plasma levels of growth hormone (GH), prolactin (PRL) and adrenocorticotropin (ACTH) were normal. When she was 23, symptoms and signs of acromegaly and subsequently galactorrhea-amenorrhea had developed. Plasma GH and PRL were increased and she was followed up by the administration of bromocriptine (2.5mg-12.5mg/day, p.o.). However the plasma GH level had been increasing gradually. On admission, plasma GH and PRL were high (19.5μg/L, 61.0μg/L, respectively) and increased in response to thyrotropin releasing hormone (TRH, 500μg iv). An intrasella mass, which had been detected when she was 17, had become enlarged and was removed by Hardy's operation. Microscopically, the resected tumor was an eosinophilic adenoma. Immunohistochemical studies showed GH, PRL and ACTH positive cells localized in the tumor. Immunoultrastructural analysis of the tumor confirmed that GH, PRL and ACTH were present in secretory granules and Golgi apparatus in the tumor cells. The patient was a rare case of acromegaly with hyperprolactinemia developed after bilateral adrenalectomy of fishing's syndrome due to adrenocortical nodular hyperplasia, all of which manifestations may be caused by a GH, PRL and ACTH secreting pituitary adenoma.

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The number of Na-K ATPase units in erythrocytes (RBC) was determined by the maximal ouabain binding assay in 25 normal subjects and patients with hyperthyroidism (n=29), hypothyroidism (8), chronic renal failure (CRF, 19) and with neoplastic disorders (NP, 12). The activity of the pump units was also assessed by measuring ouabain-sensitive ⁸⁶Rb uptake in some of these subjects. In addition, it was determined in mononuclear cells in normal controls and patients with hyper- and hypothyroidism and CRF. Significant diminution of the number of the RBC pump units was found in hyperthyroidism, while it was increased in hypothyroidism. The binding (O) of old RBC was significantly lower than that (Y) of young RBC and a striking correlation was observed between the % reduction rate ((Y-O)/Y) of the binding and the serum T₄ level in hyperthyroidism (r=0.85, P<0.02). No difference was observed in pump units of mononuclear cells in normal and hyper- and hypothyroidism. It is suggested that the thyroid hormone- mediated disappearance of the pump units in RBC may play a role in reducing the number of pump units in RBC in hyperthyroidism. The ratio of RBC ⁸⁶Rb uptake to the number of the pump units in the same cell (U/B) bore a significant relation to serum T₃(r=0.48, P<0.05) and T₄ (r=0.49, P<0.05) indicating that the U/B is a useful index for the peripheral metabolic status. In CRF patients with low T₃ levels, bindings were increased but those in NP with low T₃ was almost normal. The increased bindings were observed in NP patients with normal T₃. These findings suggest that the metabolic status in CRF and NP patients could not be always assessed by the number of the ouabain sensitive Na-K ATPase pump units. However, the U/B was almost normal in all patients with CRF and NP, suggesting that pump activity as a measure of oxygen consumption and thermogenesis may not be in the hypothyroid state in patients with nonthyroidal illnesses.

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We report a male who exhibited the Landry-Guillain-Barré syndrome and hypercalcemia. He initially exhibited normocalcemia, followed by hypercalcemia which developed during tetraplegi a and the recovering phase of the syndrome. The administration of prednisolone, saline, calcitonin, etidronate, and indomethacin failed to normalize the serum calcium level. Since, with mobilization, the serum calcium level gradually became normal, the calcium abnormality was misdiagnosed as immobilization hypercalcemia. However, among 6 different parathyroid hormone (PTH) assays used, including a two-site immunoradiometric assay, only a mid-region specific PTH (mPTH) assay showed high levels in both hypercalcemic and normocalcemic periods, and a high level of mPTH was not suppressed by calcium infusion in the normocalcemic period. Neck exploration disclosed a parathyroid adenoma weighed 100mg. This case illustrates the hypercalcemia-inducing effect of immobilization on mild type primary hyperparathyroidism. A high level mPTH assay, its unsuppressibility by the calcium infusion test, and ineffectiveness of oral etidronate for hypercalcemia were valuable in differentiating hypercalcemia due to primary hyperparathyroidism from that resulting solely from prolonged immobilization.

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We investigated the relationship between plasma insulin-like growth factor I (IGF-I) levels and the body mass index (BMI) in 558 healthy adults (238 males and 320 females), aged 21-80yrs. The subjects were divided into three groups based on the BMI: 1) underweight group (BMI<20, n=145), 2) normal group (BMI 20-25, n=179) and 3) overweight group (BMI>25, n=234). Blood samples were obtained after overnight fasting and plasma IGF-I concentrations were measured by specific radioimmunoassay after acid-ethanol extraction. Plasma IGF-I levels declined with age in each group and the regression lines were parallel. In both sexes of each decade, mean plasma IGF-I values were lower in the underweight and overweight groups than in the normal group. Age- and sex-matched analysis revealed that plasma IGF-I values were positively correlated with BMI under 25, whereas plasma IGF-I was reversely correlated with BMI beyond 27. These findings suggest that the plasma IGF-I levels are influenced by body composition independently of aging in adults, aged 21-80yrs.

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A 62-year-old patient with non-insulin dependent diabetes (NIDDM) was admitted to our hospital for blood pressure control. He had been treated with angiotensin converting enzyme inhibitor (ACEI) for 7 years and showed marked hypokalemia with increased urinary potassium excretion. Hormonal examination revealed a normal plasma aldosterone concentration and increased plasma renin activity (PRA, 13.4ng/ml/h), so potassium losing nephropathy was suspected. After discontinuation of the ACEI, PRA decreased to normal. An adrenal adenoma was found on abdominal magnetic resonance imaging (MRI) and adrenalectomy was performed to confirm aldosterone producing adenoma (APA). Although ACEIs are said not to alter PRA in APA, this drug was primarily responsible for the increased PRA in this case. This is a rare case of APA, which showed markedly increased PRA during ACEI treatment.

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Monensin is a carboxylic ionophore which perturbs the structure and function of the Golgi apparatus and lysosomes. In the present study, we investigated the functional significance of these organella in the growth factor-mediated cell proliferation in cultured human thyroid cells from normal and Graves' disease. DNA synthesis was estimated by [³H]-thymidine uptake and flow cytometric analysis. Monensin inhibited both [³H]-thymidine uptake in a dose-dependent manner and the transition of G₁ to S phase determined by flow cytometric analysis. Monensin partially blocked the effect of bovine TSH in normal thyroid cells. [³H]-thymidine uptake was suppressed to 56.7±37.3% of the control value with bTSH and monensin, but it was still higher than those with monensin alone (21.9±15.0% of the control). The percentage of cells in the S phase was also increased from 7.64±1.91% with monensin alone to 11.54±2.82% with bTSH at t=24h. Forskolin or 12-O-tetradecanoylphorbol 13-acetate (TPA) could not mimic the action of TSH. On the other hand, insulin and EGF most effectively counteracted monensin-induced inhibition of DNA synthesis in Graves' thyroid cells. [³H]-thymidine uptake was not completely inhibited, being 73.5±24.0% with EGF, 105.0±25.4% with insulin, and 49.2±6.6% with monensin alone, respectively. The percentage of cells in the S phase also increased from 8.31±2.61% with monensin alone to 11.25±4.27% with EGF and 12.86±3.12% with insulin. In conclusion, the functional maintenance of the Golgi apparatus and lysosomes is necessary for DNA synthesis in both normal and Graves' thyroid cells, in which bTSH, insulin, and EGF might be differently involved in the regulation of DNA synthesis.

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To investigate the efficacy of intravenous methylprednisolone pulse therapy on Graves' ophthalmopathy (GO), fifteen patients with severe GO were treated with large dose intravenous methylprednisolone (at a daily dosage of 1g for 3 successive days). This treatment was repeated 3-5 times for 3-5 weeks. They were monitored before, 2 weeks after and 6 months after therapy by ophthalmological assessment, orbital magnetic resonance imaging (MRI), and by measuring serum antibodies to rat eye muscle (EMAB) in an enzyme linked immunosorbent assay and peripheral blood lymphocyte subsets by flow cytometry. Diplopia and periorbital edema markedly improved after treatment in 9 patients. Mean proptosis values and intraocular pressure measurements significantly decreased after pulse therapy. In 12 patients enlarged eye muscles significantly reduced in size after treatment, as determined by MRI. The overall ophthalmopathy index was improved from 4.8±2.4 to 2.5±1.6 at the end of pulse therapy (P<0.01) and 2.4±1.5 six months after therapy (P<0.01). Serum EMAB were detected in 8 out of 10 patients tested and their level significantly decreased after pulse therapy (from 3.3±1.4 to 2.5±1.2, P<0.01). A significant increase in peripheral blood CD4⁺CD45RA⁺ cells was observed after pulse therapy. Increased numbers of CD11^-CD8⁺ cells and decreased numbers of CD11⁺CD8⁺⁺ cells were found prior to treatment and were normalized after pulse therapy. Our study indicates that methylprednisolone pulse therapy can be considered as a choice for the treatment of GO. The improvement in eye muscle involvement in these patients may be due to the effects of infused methylprednisolone on both humoral and cellular immune functions.

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Ovine luteinizing hormone (oLH), a pituitary hormone with sulfated asparagine-linked oligosaccharides, was examined with regard to how its isoforms having different isoelectric points (pIs) modulate in vivo biological activity. oLH was separated into five fractions by means of an isoelectric focusing (IEF) column, i.e., IEF fr 1 (pI>10.78), IEF fr 2 (pI 10.78-10.26), IEF fr 3 (pI 10.22-10.07), IEF fr 4 (pI 9.99-9.84), and IEF fr 5 (pI 9.76-9.48). Of these, we studied the three major fractions, IEF fr 2, 3 and 4. The binding of ¹²⁵I-oLH to rat testis homogenates was inhibited most strongly by IEF fr 4, followed by 2 and 3. The ability to stimulate cyclic AMP release from dispersed rat Leydig cells followed the same sequence. After bolus injections of IEF fractions, the plasma immunoreactive oLH levels of male rabbits were measured. IEF fr 4 and 3 had longer plasma half-lives than 2. As expected, plasma testosterone levels after injections increased with the additive effects of in vitro activity and the rate of clearance from the circulation, i.e., IEF fr 4>3=2. In vivo renotropic activity, ³H-thymidine incorporation into the renal DNA of hypophysectomized, castrated rats, was found only in IEF fr 4. Our study indicates that an oLH isoform with a lower alkali pI had stronger LH receptor binding and in vitro bioactivity and a longer plasma half-life, resulting in stronger in vivo bioactivity.

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To evaluate the usefulness of monitoring serum sialic acid (SA) levels for diagnosis and follow-up of subacute granulomatous thyroiditis (SAT), 43 patients were studied at our clinic. In the acute phase of the disease their SA levels averaged 104.9±19.7mg/dl (normal 44-69mg/dl). In the recovery phase SA levels returned to a range of 60.5±6.9mg/dl. However, an increase in SA (87.4±18.2 mg/dl) was detected at the time of recurrence in 14 patients. In 29 non-recurrent patients, serum SA gradually reduced during the course of therapy and normalized in all patients by the time glucocorticoid therapy was discontinued. Thyroglobulin (Tg) and the erythrocyte sedimentation rate (ESR), however, had normalized in only half the cases even at the time of cessation of therapy (Tg 5/11, ESR 4/8). C-reactive protein (CRP) returned to negative in most patients (19/24) only one week after initiation of the therapy. These results suggested that the monitoring of SA levels can be a useful tool in diagnosis and follow-up of SAT.

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The present study was performed to clarify changes in plasma levels of 19-hydroxyandrostenedione (19-OH-AD), an amplifier of aldosterone and a possible hypertensinogenic steroid, during several tests for the renin-angiotensin system in 20 patients with aldosterone-producing adenoma (APA) and to determine whether 19-OH-AD participates in the etiology of the hypertension in this disorder. Basal plasma 19-OH-AD levels in patients with APA were significantly lower than those in 50 normal subjects, and correlated positively with basal plasma cortisol levels (r=0.45, P<0.05). Plasma 19-OH-AD levels were not changed significantly by 2-h standing during which plasma renin activity (PRA) remained suppressed. With 40mg iv furosemide plus 2-h standing during which PRA remained suppressed, plasma 19-OH-AD levels increased significantly with a concomitant significant increase in plasma cortisol. With dexamethasone pretreatment, however, such positive responses of plasma 19-OH-AD and cortisol disappeared. After the removal of the APA with the adjacent adrenal tissue, PRA and plasma aldosterone concentrations became normal or low-normal, but plasma 19-OH-AD and cortisol did not change as compared with the preoperative levels. There were no significant correlations between basal plasma 19-OH-AD levels and mean blood pressure either before or after the adrenal operation. These findings suggested that 1) the secretion of 19-OH-AD in patients with APA is reduced due to the chronically suppressed renin-angiotensin system, 2) but is still concomitantly under the control of the ACTH-adrenal axis and 3) 19-OH-AD may, at least, not play an important causative role in the hypertension commonly observed in patients with APA.

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The effect of adrenodemedullation (ADMX) on insulin action was examined in anesthetized rats by means of a three-step euglycemic clamp procedure (insulin infusion rate: 0, 6.0 and 30.0 mU•kgBW^-1•min^-1) combined with a microdialysis technique in skeletal muscle and adipose tissue. The dialysate lactate levels in the above tissues increased in parallel with the plasma lactate levels during the sequential euglycemic clamp. In the euglycemic clamp, the glucose infusion rate (GIR) was significantly (P<0.01) higher in ADMX rats (13.41±0.82mg•kgBW^-1•min^-1) than in SHAM rats (10.21±0.87 mg•kgBW^-1•min^-1) during the 6.0-mU•kgBW^-1•min^-1 insulin infusion, and the lack of a ignificant difference between ADMX and SHAM rats was observed during the 30-mU•kgBW^-1•min^-1 insulin infusion. In skeletal muscle, the concentration of lactate in dialysate was significantly higher in ADMX rats (9.29±1.01mg/dl) than in SHAM rats (6.22±0.47mg/dl) (P<0.05) at an insulin infusion rate of 6.0mU•kgBW^-1•min^-1. In adipose tissue, no significant difference in dialysate lactate levels was found between ADMX and SHAM rats at any insulin infusion rate. These results suggest that 1) it is possible to determine insulin action in skeletal muscle and adipose tissue in vivo by using the microdialysis technique, that 2) ADMX appears to result in a significant increase in insulin sensitivity, and that 3) lactate formation increased in skeletal muscle, but not in adipose tissue.

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Late-onset congenital adrenal hyperplasia due to 3β-hydroxysteroid dehydrogenase deficiency has been reported with increasing frequency, but only a few adult women have been found to have this disorder in Japan. We report a 26-year-old Japanese hirsute woman with partial 3β-hydroxysteroid dehydrogenase deficiency. The diagnosis was based on significantly increased ratios of 17-hydroxypregnenolone to 17-hydroxyprogesterone and of dehydroepiandrosterone to androstenedione after administration of ACTH. Hirsutism improved with the administration of dexamethasone (0.5mg) every evening. Since routine assay of Δ⁵-steroid metabolites has become available, the incidence of this disorder will increase. Diagnostic effort should be attempted since the disorder is treatable with low-dose dexamethasone.

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Insulin-like growth factor II (IGF-II) in serum and tumor extracts from five patients with non-islet-cell tumor hypoglycemia (NICTH) has been characterized. These tumors contained large quantities of IGF-II (2.4-14.2μg/g tissues). The serum IGF-II levels in four of five patients were a little high and the serum IGF-I levels in five patients were low. The serum IGF-II/IGF-I ratios in these patients ranged from 24.1 to 64.2, and the values were significantly greater than those in normal subjects (1.7-7.1). When the sera were gel-filtered on a Sephacryl S-200 column under neutral conditions, the proportion of the free form of IGF-II was not increased. However, in four of five patients, an abnormal IGF-II-IGF binding protein complex was found. When serum IGF binding proteins (IGFBPs) were analyzed by Western ligand blotting, serum IGFBP-2 increased in these patients. When the tumor extracts and sera were gel-filtered on a Biogel P-60 column under acidic conditions, the majority of IGF-II in these sera was a big form of IGF-II. As compared to authentic IGF-II, insulin receptor reactivities and IGF-II receptor reactivities of tumor extracted IGF-II increased in two of three patients. These data indicate that in patients with NICTH, heterogenous IGF-II is produced in respect of size and bioactivities, and that the characteristics of IGF binding protein are altered. Thus, to find IGF-II producing tumors among extrapancreatic tumors associated with hypoglycemia, the quality of IGF-II as well as the quantity should be studied.

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A 65-year-old female patient was admitted with complaining chiefly of lower back pains and arthralgia in the bilateral knee joints of 10-years duration. The serum calcium concentration was normal or only slightly increased, whereas the serum intact PTH and 1, 25-dihydroxyvitamin D concentrations were substantially increased. Serum phosphate and 25-hydroxyvitamin D concentrations were decreased. Renal function was normal. Serum alkaline phosphatase activity, the osteocalcin concentration and urinary hydroxyproline excretion were markedly increased. Bone X-ray examination showed severe osteopenia and bone biopsy revealed hyperosteoidosis without tetracycline deposition, consistent with osteomalacia. A parathyroid adenoma was demonstrated by echography and CT-scan. Surgical exploration of the neck revealed a chief cell adenoma behind the right upper pole of the thyroid gland. After parathyroidectomy, all the abnormal biochemical data gradually normalized and the patient has been doing well without any symptoms for the last 13 months. These clinical data suggest that osteomalacia of the patient was probably induced by hypophosphatemia of prolonged duration. When hypercalcemia is not evident in a patient with primary hyperparathyroidism, in whom serum alkaline phosphatase and intact PTH levels are inappropriately increased, osteomalacia should be taken into consideration.

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Thyroxine-binding globulin (TBG) is the major transport protein of thyroid hormones in human serum. In this communication, we present a sequence abnormality of the TBG-gene in a Japanese family manifesting partial TBG deficiency (TBG-PDJ). The propositus was a male with a reduced concentration of TBG (3.2μg/ml). Thyroid function tests suggested that the inheritance of this TBG abnormality was X-linked. The TBG exhibited increased heat-lability compared with the common type TBG (TBG-C). The isoelectric focusing pattern of this TBG molecule was indistinguishable from TBG-C. Genomic DNAs from white blood cells of four members of a TBG-PDJ family were subjected to polymerase chain reaction (PCR), and the products were sequenced. The sequencing of the entire coding exons and exon/intron junctions of TBG allele of the propositus revealed a single nucleotide substitution: CCT (proline) to CTT (leucine) at amino acid 363 of the TBG-C. The heterozygosity as revealed by the direct sequencing of the PCR product correlated with the TBG concentration in serum. The proline to leucine substitution may cause a change in the TBG tertiary structure and result in decreased heat stability, resulting in decreased TBG levels in the affected subjects.

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It has been suggested that pit-1 protein may play a role in the differentiation of the anterior pituitary cells. The present Immunohistochemical studies were designed to elucidate the relationship between functional differentiation of pituitary adenoma and expression of pit-1 protein in human (h) GRF transgenic mice. Pituitaries from a 10 month old and a 6 month old transgenic mice were fixed in 4% paraformaldehyde and embedded in paraffin. The indirect immunoperoxidase method was performed using antibodies against hGRF, GH, PRL, ACTH, α subunit (SU), FSHβ SU, LHβ SU, TSHβ SU, and pit-1 protein. Immunohistochemical double staining was performed at light and electron microscopic levels. The pituitary glands of hGRF transgenic mice (both 10 month and 6 month old) demonstrated diffuse hyperplasia of GH positive cells with coexpression of hGRF within the same cells. There were also scattered cells which were positive for other hormones and hormone subunits in the hyperplastic pituitary. Three discrete nodules were found in the pituitary gland of a 10 month old hGRF transgenic mouse and were identified as adenomas. These adenomas were composed of enlarged round cells which were positive only for GH, hGRF, PRL and TSHβ SU. Pit-1 protein was intensely expressed in the nuclei of the adenoma cells. These results suggest the existence of an autocrine mechanism by hGRF in the formation of somato-lacto-thyrotroph adenoma via constitutive pit-1 expression.

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Thyroid peroxidase (TPO) was purified from each thyroid of 10 patients (experiment 1) and 4 patients (experiment 2) with toxic diffuse goiter by a simplified method with monoclonal anti-TPO antibody-assisted immunoaffinity column chromatography. The final preparations were used to measure the heme concentration based on the cyanide difference spectrum, and to determine the Km and k_cat values from double reciprocal plots in the assay employing guaiacol and iodide as the second substrates. The heme-based specific activities of TPO purified from thyroids in experiment 1 were higher than those in experiment 2, which were probably impaired by freezing-thawing, and those of porcine TPO previously reported. There were some differences in the kinetic properties between experiment 1 and experiment 2, but the individual differences within each group were relatively small, the values for CV (=SD/mean) being 0.16-0.48.

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Thyroid peroxidase (TPO) is an essential enzyme involved in thyroid hormone synthesis and is closely related to the microsomal antigen which is the target of thyroid microsomal antibody. There have been several reports on direct inhibition of peroxidase activity by thyroid microsomal antibody. We prepared a mini organ culture of thyroid glands obtained at operation, and investigated the localization of thyroid peroxidase activity in follicular cells proliferated around the thyroid tissue blocks by electron microscopy. The development of microvilli containing TPO activity on the cell surface facing the culture medium was observed when normal thyroid tissue or Graves' thyroid tissue was incubated with TSH but in the TSH-free group the development of microvilli was poor and TPO activity was very much decreased. After the addition of serum positive for thyroid microsomal antibody, the TPO activity of the microvilli was retained in 4/6 tissue samples, but it disappeared in 2 cases. Our findings suggested that thyroid peroxidase activity is regulated by thyroid stimulating substances such as TSH and by TPO in tissue.

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To investigate the peripheral metabolic status during normal pregnancy, we measured the number of erythrocyte Na, K-ATPase units as well as the cation transport activity of the pump from 32 normal pregnant women and 12 normal controls. The number of pump units determined by maximal ouabain binding to erythrocyte in normal pregnancy was significantly higher than that in normal controls (mean±SEM: 0.52±0.03 vs. 0.39±0.04pmol/10⁹RBC, P<0.05). The total cation transport activity of the pump measured by ⁸⁶Rb uptake also significantly increased during pregnancy (98.9±6.4 vs. 73.1±5.4nmol/10⁹ RBC, P<0.01). However, the mean cation transport activity per pump unit, which was presumed to be an indicator of the peripheral metabolic status, was unchanged in any of three trimesters when compared with that in normal controls. Serum FT₄ levels measured by two different methods were significantly lower in the third trimester than in the first trimester (P<0.01). In conclusion, erythrocyte Na, K-ATPase activity per pump unit is normal in pregnant women, suggesting that the peripheral metabolic status in pregnancy seems to be normal. Increases in both the number and function of the pump may be influenced by factors other than thyroid function.

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