[Report of immunoassay control surveys conducted in the past 30 years by the Subcommittee for Radioisotope in vitro Test, the Medical Science and Pharmaceutical Committee, and the Japan Radioisotope Association].

Immunoassay control surveys, were conducted by the Subcommittee for Radioisotope in vitro Test, the Medical Science and Pharmaceutical Committee, and the Japan Radioisotope Association, between 1978 to 2008. A total of 40 analytes for 26 hormones, 14 tumor markers and pharmaceutical drugs were investigated in participating facilities. In the first immunoassay control survey in 1978, samples were measured using only RI kits, however, non-RI kits increased gradually during the next 30 years. In the 30th immunoassay control survey, more than 90% samples were measured using non-RI kits. Coefficient variation (CV) of intra-kits has been decreasing yearly in all analytes for hormones as well as tumor markers. However, improvement of CV in inter-kits has not been seen in the past 30 years by a lack of international standards, although there has been continuous effort over the years for the standardization of immunoassay. Growth hormone (GH) deficiency has been diagnosed using various loading tests. However, the clinical diagnosis varies according to the GH kit used. Standardization for GH measurement has been possible by using recombinant GH as the standard among commercial GH kits. The diagnosis of subclinical Cushing's syndrome also varies according to the cortisol kits being used. Candidate reference measurement procedure and low level cortisol standards have been developed by the Biomedical Standard Section, of the National Metrology Institute of Japan. Standardization of measurement is necessary for improvement of immunoassay.

Identification and quantification of plasma free salusin-ß, an endogenous parasympathomimetic peptide.

Salusin-ß is an endogenous parasympathomimetic proatherosclerotic peptide. Salusin-ß was initially predicted from bioinformatic analyses and later immunologically detected in human biofluids. However, elucidation of salusin-ß bioactivity has faced enormous challenges because of its unique physicochemical characteristics that cause it to strongly adhere to laboratory apparatus materials. In the strictest sense, the discovery of bioactive peptides is not complete until their exact native sequences have been confirmed in the peripheral circulation. In this study, we determined the plasma molecular form and levels of free salusin-ß to determine its pathophysiological significance. Ultra-high-yield enrichment and preseparation of non-tryptic human plasma was followed by LC-MS/MS, and full-length salusin-ß and seven different endogenous fragment sequences were identified. We established a new ELISA that specifically detects plasma free salusin-ß without cross-reacting with any of its identified endogenous fragments. Free salusin-ß levels exhibited a profound early morning nadir and rapidly decreased in response to parasympathetic nervous augmentation. Our technical advance in plasma native peptide analysis successfully identified a hard-to-detect bioactive peptide, salusin-ß, together with its formerly unrecognized fragments, and further suggests that conventional immunological measurements of target peptides may not be fully representative.

Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

BACKGROUND: We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. METHODS: We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. RESULTS: A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. CONCLUSIONS: Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis.

Metastatic Pancreatic Neuroendocrine Tumor that Progressed to Ectopic Adrenocorticotropic Hormone (ACTH) Syndrome with Growth Hormone-releasing Hormone (GHRH) Production.

The patient was a 61-year-old woman who had a well-differentiated pancreatic neuroendocrine tumor (PNET) with lymph node metastasis. After 15 months of octreotide treatment, glucose control deteriorated and pigmentation of the tongue and moon face developed, leading to the diagnosis of ectopic adrenocorticotropic hormone (ACTH) syndrome. An abnormal secretion of growth hormone (GH) was identified, and the plasma growth hormone-releasing hormone (GHRH) level was elevated. A tumor biopsy specimen positively immunostained for ACTH and GHRH. Ectopic hormone secretion seems to have evolved along with the progression of the PNET.

Isolated Adrenocorticotropin Deficiency Concomitant with Graves' Disease: A Case Report and Literature Review.

A 73-year-old Japanese woman with untreated Graves' hyperthyroidism developed glucocorticoid-induced adrenal insufficiency (AI) after a supraphysiological dose of prednisolone therapy for bronchial asthma. Days later, she had high plasma adrenocorticotropic hormone (ACTH) levels and was expected to recover from glucocorticoid-induced AI. Her plasma ACTH levels remained high over 3 months during a physiological dose of hydrocortisone replacement. However, she suffered a further decrease in her serum cortisol level and was diagnosed with isolated adrenocorticotropin deficiency (IAD), in which bioinactive ACTH likely caused the high ACTH value. IAD should be considered as an unusual disorder associated with Graves' disease, especially in older patients.

Acute Exacerbation of Idiopathic Pulmonary Fibrosis Following Treatment for Cushing's Syndrome.

A 64-year-old Japanese man with mild reticular shadows in both lungs developed a lung tumor causing ectopic Cushing's syndrome. He was prescribed an adrenal inhibitor, which controlled his hypercortisolemia. However, he developed acute exacerbation of idiopathic pulmonary fibrosis (IPF) and died within weeks. Previous studies have suggested a dosage reduction of corticosteroids for IPF as a triggering event for acute exacerbation. The present case suggests that IPF coexisting with Cushing's syndrome may have been exacerbated after the correction of hypercortisolemia. Therefore, close monitoring of cortisol levels along with the clinical course of IPF is required in similar cases that require the correction of hypercortisolemia.

Human chorionic gonadotropin detection in cerebrospinal fluid of patients with a germinoma and its prognostic significance: assessment by using a highly sensitive enzyme immunoassay.

OBJECTIVE Human chorionic gonadotropin (HCG) can be detected in a certain population of patients with a germinoma, but the frequency of germinoma HCG secretion and the prognostic value of HCG in the CSF are unknown. METHODS The authors measured HCG levels in sera and CSF in patients with a histologically confirmed germinoma by using a highly sensitive assay known as an immune complex transfer enzyme immunoassay (EIA), which is more than 100 times as sensitive as the conventional method, and they analyzed the correlation between HCG levels and the prognoses of patients with a germinoma. RESULTS HCG levels in sera and CSF of 35 patients with a germinoma were examined with the immune complex transfer EIA. The median CSF HCG levels in patients with a germinoma during the pretreatment and posttreatment evaluations were 192.5 pg/ml (range 1.2-13,116.5 pg/ml) and 18.7 pg/ml (1.2-283.9 pg/ml), respectively. Before treatment, the CSF HCG level was greater than the cutoff value in 85.7% of the patients with a germinoma. The authors compared survival rates among the patients by using a CSF HCG cutoff level of 1000 pg/ml, and the difference was statistically significant between the groups (p = 0.029, log-rank test). CONCLUSIONS Results of this study demonstrate that most germinomas secrete HCG. Patients with a germinoma that secretes higher amounts of HCG in their CSF experienced recurrence more frequently than those with lower CSF HCG levels.

Pancreatic solitary fibrous tumor causing ectopic adrenocorticotropic hormone syndrome.

Solitary fibrous tumors occasionally present with hypoglycemia because of the excessive release of insulin-like growth factor II. We report the first case of pancreatic solitary fibrous tumor causing ectopic adrenocorticotropic hormone syndrome. An 82-year-old Japanese man presented with lower limb edema, uncontrolled hypertension, hypokalemia, and baseline hypercortisolism. Distal pancreatectomy was performed after the clinical diagnosis of a neuroendocrine tumor with ectopic secretion of adrenocorticotropic hormone. On histological examination, the tumor showed spindle cells in a fascicular arrangement. The diagnosis of the solitary fibrous tumor was confirmed by the identification of the NAB2-STAT6 fusion gene and positive immuno-histochemical staining for STAT6 and CD34. Using quantitative real-time polymerase chain reaction, mRNA that encoded proopiomelanocortin, precursor of adrenocorticotropic hormone, was detected. Proopiomelanocortin production through the demethylation of the promoter region Domain IV was detected. Pancreatic solitary fibrous tumors represent a new cause of ectopic adrenocorticotropic hormone syndrome.

Aging-related changes in in vivo release of growth hormone-releasing hormone and somatostatin from the stalk-median eminence in female rhesus monkeys (Macaca mulatta).

GH release decreases with aging in primates. However, it is unclear whether the age-related decrease in GH release is due to a decrease in stimulatory GHRH or an increase in inhibitory somatostatin (SS) from the hypothalamus. In the present study, we measured the release of GHRH and SS in the stalk-median eminence of conscious aged (n = 7, 27.0 +/- 0.7 yr old) and young adult female monkeys (n = 12, 5.0 +/- 0.3 yr old) using the push-pull perfusion method. Mean GHRH levels during morning (0600-1200 h) and evening (1800-2400 h) in aged monkeys were 3- to 4-fold lower than in young monkeys. Pulse analysis indicated that pulse frequency, pulse amplitude, and baseline GHRH release in aged monkeys were much lower than in young adults. In contrast, mean SS levels in aged monkeys during mornings and evenings were 2-fold higher than in young monkeys. Pulse analyses indicated that amplitude and baseline levels of SS were significantly higher in aged monkeys than in young adults. There were no significant changes in the pulse frequency of SS release. Therefore, the aging-related decrease in GH release is due to a substantial decrease in GHRH release and an increase in SS release from the hypothalamus.

Plasma levels of intact and degraded ghrelin and their responses to glucose infusion in anorexia nervosa.

Octanoylated ghrelin (1-28) (intact ghrelin) is rapidly and easily degraded to desoctanoyl forms or smaller fragments (degraded ghrelin). Plasma levels of intact and degraded ghrelin were examined in 30 patients with anorexia nervosa (AN) (body mass index, 8.81-22.4 kg/m(2)) and 16 age-matched healthy women using several assay methods. Plasma levels of ghrelin measured using immunocomplex transfer-enzyme immunoassay, which specifically detects intact ghrelin, were lower in AN than controls. Plasma ghrelin levels in AN measured using the active ghrelin ELISA kit, which is advertised as specifically detecting intact ghrelin, did not differ significantly from controls. Plasma levels of desoctanoyl ghrelin using the desacyl-ghrelin ELISA kit, N-terminus ghrelin using the ghrelin active RIA kit, and C-terminus ghrelin using the ghrelin total RIA kit were significantly higher in AN than controls, and displayed significant negative correlations with body mass index. Plasma levels of ghrelin determined using immunocomplex transfer-enzyme immunoassay or active ghrelin ELISA during iv glucose infusion were suppressed in both AN and controls, whereas plasma levels of degraded ghrelin levels were not significantly decreased in AN. Plasma levels of intact ghrelin are therefore not higher in AN than controls, whereas degraded forms of ghrelin are elevated in AN. Rapid suppression of plasma intact ghrelin, but not degraded ghrelin, occurs in AN in response to glucose infusion. The profiles of intact and degraded forms of ghrelin in plasma of AN patients differ from those of healthy women.

Development of an ultra-sensitive enzyme immunoassay for human insulin autoantibodies.

OBJECTIVES: We developed an ultrasensitive enzyme immunoassay (ICT-EIA) for insulin autoantibody (IAA) measurements to better understand the pathophysiology of diabetes. DESIGN AND METHODS: We developed ICT-EIA for IAA and measured IAA in 24 patients with type 1 diabetes, 30 patients with type 2 diabetes, 30 patients with methimazole-treated Graves' disease, 20 patients with Hashimoto's disease, 9 patients with hyperinsulinemia, and 73 healthy control subjects. RESULTS: The conventional ELISA identified 3 patients with type 1 diabetes and 2 patients with type 2 diabetes as IAA positive, whereas 15 patients with type 1 diabetes, 7 patients with type 2 diabetes, and 4 patients with methimazole-treated Graves' disease were identified as IAA positive using ICT-EIA. CONCLUSIONS: The ICT-EIA is an ultrasensitive and specific assay for IAA, and its use may provide a better understanding of the role of IAA in diabetes onset and progression.

A mechanism of acquiring temozolomide resistance during transformation of atypical prolactinoma into prolactin-producing pituitary carcinoma: case report.

BACKGROUND AND IMPORTANCE: The case presented here describes the clinical evolution of a pituitary carcinoma from an atypical prolactinoma after temozolomide (TMZ) treatment. The mechanism of acquisition of TMZ resistance was analyzed. CLINICAL PRESENTATION: A 60-year-old woman with atypical prolactinoma had been treated for 7 years with multiple therapies, including dopamine agonists, surgical intervention (5 times), conventional radiotherapy, and radiosurgery. The patient deteriorated as a result of tumor enlargement. Ten cycles of TMZ therapy, 200 mg/m for 5 days every 4 weeks, improved the patient's performance status and caused tumor shrinkage. Six months after discontinuation of TMZ, the tumor progressed into pituitary carcinoma with tumor regrowth and intraventricular dissemination. TMZ therapy was ineffective this time. A sixth surgery and salvage chemotherapy failed to improve the patient's condition, and she died 9 years after the first diagnosis. Throughout the treatment course, O6-methyl-guanine-DNA methyltransferase (MGMT) was immunonegative in the tumor specimens, including the TMZ-refractory pituitary carcinoma. Mutation of p53 was identified in both the atypical prolactinoma and pituitary carcinoma. In contrast, major differences were noted for mismatch repair protein MSH6 immunostaining: Although MSH6 was diffusely immunopositive in the atypical adenoma, it became immunonegative when the tumor evolved into TMZ-refractory pituitary carcinoma. CONCLUSION: Loss of MSH6 occurred during the progression from an atypical prolactinoma to a pituitary carcinoma, which may have caused resistance to TMZ treatment. This case suggests that preserving MSH6 function is essential for responsiveness to TMZ treatment in MGMT-negative and p53-mutated atypical pituitary adenoma or pituitary carcinoma.

Skull base metastasis from follicular thyroid carcinoma -two case reports-.

A 58-year-old woman and a 71-year-old woman presented with extremely rare skull base metastases from follicular thyroid carcinoma (FTC). Surgical removal and external radiotherapy were performed followed by iodine-131 ((131)I) brachytherapy and thyroid hormone administration. The metastatic tumors in the skull base were well controlled. Treatment for skull base metastasis from FTC includes surgical debulking of the metastatic lesion, as well as complete resection of the thyroid gland, followed by internal irradiation with (131)I, external irradiation, and administration of thyroid hormone to prevent tumor growth by suppression of endogenous thyroid-stimulating hormone. Skull base metastases may be the initial clinical presentation of FTC, with silent primary sites. The possibility of skull base metastasis from FTC should be considered in patients with clinical symptoms of cranial nerve dysfunction and radiological findings of bone destruction.

A case of acquired deficiency of pituitary GH, PRL and TSH, associated with type 1 diabetes mellitus.

A 75-year-old male showed combined anterior pituitary hormone deficiency (CPHD). Basal and TRH-stimulated PRL levels were undetectable. Basal and GRH-stimulated GH levels were very low, and could barely be measured by means of an ultrasensitive enzyme immunoassay. In addition, basal TSH levels were under the normal limit, and TRH-stimulated TSH secretions were impaired. On the other hand, the secretions of ACTH, LH and FSH remained intact. There was no mutation of Pit-1 gene in this patient, and immunohistochemical studies using human pituitary and the patient's serum showed no positive staining. The HLA types frequently detected in lymphocytic hypophysitis were recognized, supporting the view that the CPHD in this case may be caused by lymphocytic hypophysitis, although magnetic resonance imaging of the pituitary gland showed no specific findings. Interestingly, a high titer of anti-glutamic acid decarboxylase antibody, suggested that the patient suffered from type 1 diabetes mellitus (DM). Five years ago, his thyroid function was normal and the treatment of DM with oral hypoglycemic agent was effective, indicating that the onset of both diseases at least occurred within the last half decade. We report here a rare case of SPIDDM with CPHD which might be caused by lymphocytic hypophysitis.