Ryuichi Yamasaki

Production and secretion of immunoreactive growth hormone‐releasing factor by pheochromocytomas

The production and secretion of immunoreactive growth hormone‐releasing factor (IR‐GRF) by pheochromocytomas were examined immunohistochemically and immunochemically. GRF‐immunoreactive (GRF‐IR) cells were found, although sparsely, in 2 of 13 tumors (Cases 1 and 2), while somatostatin (SRIF)‐IR cells and vasoactive intestinal peptide (VIP)‐IR cells were found in nine and five tumors, respectively. Concentrations of tissue IR‐GRF of 29.8 and 17.2 ng/g wet weight tissue, respectively, were found in two (Cases 1 and 2) of three tumors examined. These three tumors also contained IR‐SRIF at 19.5–105.5 ng/g wet weight tissue and IR‐VIP at 13.6–24.8 ng/g wet weight tissue. An increased plasma IR‐GRF concentration (30.0 pg/ml) was found in a blood sample taken from the inferior vena cava near the adrenal tumor in Case 1. This is the first report that some pheochromocytomas produce GRF and secrete it into the blood circulation.

Radioimmunoassay for activin A/EDF: Method and measurement of immunoreactive activin A/EDF levels in various biological materials

A radioimmunoassay (RIA) for the measurement of activin A, which is identical to erythroid differentiation factor (EDF), has been developed. A specific antiserum against activin A/EDF was raised in rabbits using a mixture of recombinant EDF and polyvinyl pyrrolidone. Of the compounds tested this polyclonal antibody cross-reacted only with bovine inhibin (3.2%) and human TGF-beta (4.2%). The least detectable value in this assay was 0.06 ng/tube. The within- and between-assay coefficients of variation at three different concentrations were 3.6-9.8% and 3.4-7.7%, respectively. Using this RIA, immunoreactive activin A/EDF levels in various biological fluids and tissues were examined. The dose-response curves of porcine follicular fluid and ovarian extract were parallel to the standard curve, and porcine follicular fluid contained high activin A/EDF immunoreactivity (1050 ng/ml). On gel chromatography of porcine follicular fluid, the major immunoreactivity was eluted in the same position as authentic activin A/EDF. Human placental extract and amniotic fluid had relatively high immunoreactive activin A/EDF levels (174 ng/g wet wt. and 63.9 ng/ml, respectively), but the dose-response curve of amniotic fluid was not parallel to the standard curve. Among rat tissues, the ovary showed the highest activin A/EDF immunoreactivity (163 ng/g wet wt.) much lower than that of porcine ovary (1020 ng/g wet wt.). Low immunoreactive activin A/EDF levels were detected in most parts of rat brain (8.7-14.2 ng/g wet wt.), except for the pituitary gland (70.0 ng/g wet wt.). The initial plasma half clearance time (t1/2) of exogenous activin A/EDF was 14 min in the rat and the plasma FSH concentration did not change significantly during this period. These results suggest that this RIA system has sufficient sensitivity and specificity to measure activin A/EDF concentrations in biological materials, and that the reproductive tissues are the main sources of activin A/EDF.

Immunoreactive somatostatin and calcitonin in pulmonary neuroendocrine tumor

A well‐differentiated neuroendocrine carcinoma of the lung that secreted immunoreactive somatostatin (IR‐SRIF) and IR‐calcitonin (CT) in a 72‐year‐old woman is described. The plasma concentrations of IR‐SRIF (57.5 pg/ml) and IR‐CT (340 pg/ml) before operation were significantly higher than the respective normal ranges. After resection of the tumor, the plasma CT level (105 pg/ml) decreased to within the normal range, and the SRIF level (32.7 pg/ml) also decreased, but was still abnormally higher, which suggested the presence of an unidentified remnant of the tumor. Abnormal accumulation of technetium 99m (99mTc) in the lumbar vertebrae was found 6 months after the operation, which indicated a metastatic tumor. The tissue concentrations of IR‐SRIF and IR‐CT were 103 and 94 ng/g wet weight, respectively, and SRIF‐IR tumor cells and CT‐IR tumor cells were demonstrated immunohistochemically. On gel‐filtration chromatography of the tumor tissue, two peaks of SRIF immunoreactivity were eluted in the positions of synthetic SRIF‐28 and SRIF‐14, respectively. Conversion of SRIF‐28 to SRIF‐14 was suggested from results on changes in the two IR‐SRIF components during incubation with a crude enzyme preparation extracted from the tumor tissue.

Radioimmunoassay of an analog of luteinizing hormone-releasing hormone, [D-Ser(tBu)]6des-Gly-NH210 ethylamide (Buserelin)

A sensitive and specific radioimmunoassay is described for plasma and urinary levels of [D-Ser(tBu)]6des-Gly-NH2(10) ethylamide (buserelin). No appreciable cross-reaction (less than 0.05%) was observed with LH-RH and its analogs other than buserelin fragments (1.6-45%). The sensitivity was 3 pg per tube. At buserelin concentrations of 125, 250 and 500 pg/ml, the intra- and inter-assay coefficients of variation were 7.9, 10.0 and 10.0%, and 19.0, 7.8 and 6.8% respectively. Recovery of buserelin added to plasma was quantitative (62.5 pg/ml, 101.6%; 125 pg/ml, 76.8% and 250 pg/ml, 63.4%). A dose of 5 micrograms buserelin injected subcutaneously into 5 normal male adults, reached a peak plasma level in 45 min (mean value 119.3 +/- 47.3 pg/ml) and remained detectable for at least 4 h. The half disappearance time was 118.8 +/- 26.0 min. Between 9 and 16% of the administered dose was excreted in the urine within 24 h. Buserelin could also be detected in the plasma after intranasal administration of doses of 150, 300 and 450 micrograms. There was a significant difference in the area under the curve (AUC) for plasma levels after subcutaneous injection of 5 micrograms and intranasal administration of 150 micrograms, but not between the AUC values after the three intranasal doses. These results indicate that this method for radioimmunoassay of buserelin is suitable for analyzing the pharmacokinetics and bioavailability of buserelin in man.

Radioimmunoassay of growth hormone-releasing hormone (GHRH) with a polyclonal antibody against synthetic GHRH(1–29)-Gly4-Cys-NH2: Method and clinical studies

A radioimmunoassay (RIA) for growth hormone-releasing hormone (GHRH) using a polyclonal antibody against synthetic GHRH(1-29)-Gly4-Cys-NH2 has been developed. The antiserum (RBM105) showed full cross-reactivity with GHRH-(1-44)NH2, GHRH-(1-40)OH, GHRH-(1-37)OH and GHRH-(3-44)NH2, and probably recognized the region of Ala4 to Lys12 of GHRH. Since the sensitivity of the GHRH RIA was 1.5 pg/tube, the lowest detectable plasma level was 5 ng/l when an extract of 0.3 ml of plasma per tube was used. On gelfiltration chromatography, the GHRH immunoreactivity of normal plasma was eluted in the same position as synthetic GHRH. The plasma GHRH concentration in healthy subjects was 20.5 +/- 6.5 ng/l (mean +/- SD), and in patients with hypothalamic disorders was 17.4 +/- 2.0 ng/l. In contrast, the plasma GHRH level in hemodialysis-dependent, chronic renal failure (CRF-HD) patients (38.7 +/- 13.1 ng/l) was significantly higher than normal. The acromegalic patients were 24.3 +/- 11.9 ng/l, except for one patient with ectopic GHRH syndrome (990 ng/l): his plasma GHRH level reached 7,100 ng/l during operation, and then decreased logarithmically to 70 ng/l after 6 h. Somatostatin at concentrations of 10 and 1,000 nmol/l significantly suppressed (GHRH release) from primary culture cells of the GHRH-producing tumor from 17.3 +/- 0.92 ng/2 x 10(5) cells to 9.98 +/- 3.61 and 4.32 +/- 1.01 ng/2 x 10(5) cells, respectively after 48 h. These data indicate that this GHRH RIA is useful for determining the plasma GHRH concentration in normal and diseased states and also for in vitro studies of GHRH release.

[A case of HCG-producing suprasellar germinoma with hypothalamic syndrome associated with acute renal failure].

HCG産生鞍上部胚芽腫により,尿崩症,高Na血症,視床下部性下垂体前葉機能低下症,高プロラクチン血症などの視床下部症候群を呈し,経過中に急性腎不全を合併した14才男性例を報告した. TRH・LHRH・LVP・GHRHによる4者負試験およびinsulin, arginine, L-dopaの各負荷試験を用いて,放射線照射と化学療法による治療前後の視床下部下垂体機能の変化を明らかにした.急性糸球体腎炎に起因すると思われる急性腎不全期には,尿量の減少,クレアチニンクリアランスの低下およびNa血症の改善を認めた.尿崩症に腎不全が合併した症例は未だ報告がなく,尿崩症の場合の尿量も糸球体〓過率によって強く影響をうけることが示された.