Haruhiko Saito

Immunoreactive somatostatin and vasoactive intestinal polypeptide in adrenal pheochromocytoma. An immunochemical and ultrastructural study.

An adrenal pheochromocytoma producing somatostatin (SRIF) and vasoactive intestinal polypeptide (VIP) in a 17‐year‐old boy is presented. High concentrations of immunoreactive (IR)‐SRIF were found in plasma taken from the antecubital vein (31.0–33.0 pg/ml) and the inferior caval vein near the tumor (54.6 pg/ml), but after removal of the tumor the values became normal (11.0–15.2 pg/ml). In two portions of the resected tumor, considerable but different amounts of IR‐SRIF (151.7 and 12.1 ng/g wet wt) and IR‐VIP (13.0 and 5.5 ng/g wet wt) were demonstrated with size heterogeneities. Immunohistochemically, many IR‐SRIF cells and a few IR‐VIP cells were observed, but no cell reacting with both anti‐SRIF and anti‐VIP sera was found. Electronmicroscopically, many tumor cells had catecholamine‐like granules (250–350 nm in diameter) while some others had VIP‐like granules (100–140 nm in diameter). However, no granules resembling the SRIF granules seen in the pancreatic D cells were found. This seems to be the first report of an adrenal pheochromocytoma that produces SRIF and VIP simultaneously. It provides information on the histogenesis of hormone‐producing neurogenic tumors. Cancer 52:282‐289, 1983.

Radioimmunoassay for atrial natriuretic peptide: method and results in normal subjects and patients with various diseases

A sensitive and specific radioimmunoassay for alpha-human atrial natriuretic polypeptide (alpha-hANP) was developed to determine its plasma level. Anti-alpha-hANP rabbit serum was specific for the N-terminus and ring structure of alpha-hANP, and showed no appreciable cross-reactions with other neuropeptides. The lowest level of alpha-hANP detectable by this radioimmunoassay was 4 pg per tube. The intra- and inter-assay coefficients of variation were 4.6-11.4% and 7.9-11.8%, respectively, and the recovery rates at 4 concentrations were 62.6-74.0%. The fasting plasma alpha-hANP concentration in normal subjects were 19.3 +/- 1.0 ng/l (mean +/- SE; n = 54), and there was no sex difference. The plasma alpha-hANP level in normal subjects fell significantly during water deprivation and increased significantly on infusion of hypertonic saline. The mean plasma levels of alpha-hANP were higher than normal in patients with essential hypertension, liver cirrhosis, congestive heart failure and chronic renal failure. Our results indicate that this radioimmunoassay is suitable for determining the alpha-hANP concentration in human plasma and can assess changes in pathological and physiological states.

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.

Ontogenetic appearance of immunoreactive GRF-containing neurons in the rat hypothalamus

SummaryOntogenetic development of GRF-containing neurons in the rat hypothalamus was studied employing antisera which were generated against hpGRF (1–44)NH2 and rhGRF(1–43)OH: anti-hpGRF-C and -rhGRF sera recognize the species-specific C-terminal portions of the peptides, and anti-hpGRF-MC and -N sera recognize hpGRF(27–44)NH2 and the N-terminal portion of hpGRF(1–44)NH2, respectively. The anti-hpGRF-C and-rhGRF sera stained different neuronal cell bodies, which were localized in distinct hypothalamic areas. The former serum did not stain the axonal terminals in the median eminence, but the latter stained them strongly. The antihpGRF-MC and -N sera stained neuronal cell bodies, some of which corresponded to those immunolabelled with antihpGRF-C or -rhGRF serum. The anti-rhGRF serum first demonstrated immunoreactive perikarya in the ventral-lateral border of the arcuate nucleus of 19.5-day-old fetuses that had received an intraventricular colchicine administration 24 h previously. The immunoreactive fibers were recognized first in the external layer of the median eminence of untreated fetuses on day 19.5 of gestation, and then they increased in amount with development. No immunore-active fibers, however, were found in the median eminence of colchicine-treated animals during the fetal period. It is concluded that in rats GRF may be synthesized in the perikarya on day 18.5 of gestation and conveyed to the median eminence without delay via axonal flow.

Co-storage and co-secretion of somatostatin and catecholamine in bovine adrenal medulla

Co-storage and co-secretion of somatostatin (SRIF) and catecholamine (CA) were demonstrated using bovine adrenal medulla. In the experiment of retrograde perfusion system of isolated adrenal gland, the basal concentration of immunoreactive (IR)-SRIF was 20 pg/4 ml/min, but a significant increase of the value (142 pg/4 ml/min) was observed at 1-2 min after a brief infusion of acetylcholine (10 mM) and the marked release continued for over 6 min. Similar result was obtained for CA release. On gel-filtration chromatography of a soluble lysate of chromaffin vesicles prepared by differential and discontinuous sucrose density centrifugation, IR-SRIF was separated into three components with molecular sizes corresponding to prepro-SRIF (76.8% of the total immunoreactivity), SRIF28 (14.1%) and SRIF14 (9.1%).

Immunoreactive somatostatin in catecholamine-producing extra-adrenal paraganglioma

A patient with catecholamine‐producing extra‐adrenal paraganglioma was found to have a high concentration of immunoreactive somatostatin (IR‐SRIF) in the peripheral blood plasma (47.0 pg/ml, normal; 13.3 ± 5.3 pg/ml, mean ± SD). After removal of the tumor, the plasma SRIF level fell within normal range (8.7 pg/ml). In the resected tumor, a considerable amount of IR‐SRIF (71.0 ng/g wet weight) was also detected and SRIF‐positive tumor cells were demonstrated by the immunoperoxidase technique. Chromatographic analysis of an extract of the tumor showed the presence of two IR‐SRIF components of large molecular size besides a component consistent with authentic tetradecapeptide SRIF (SRIF 14), and one of these had the same molecular size as octacosapeptide SRIF (SRIF 28). This seems to be the first report of a catecholamine‐producing extra‐adrenal paraganglioma that produces IR‐SRIF with molecular heterogeneity.

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.

No increased risk of major congenital anomalies or adverse pregnancy or neonatal outcomes following letrozole use in assisted reproductive technology.

STUDY QUESTION Does letrozole use increase the risk of major congenital anomalies and adverse pregnancy and neonatal outcomes in fresh, single-embryo transfer? SUMMARY ANSWER Letrozole significantly decreases the risk of miscarriage and does not increase the risk of major congenital anomalies or adverse pregnancy or neonatal outcomes compared with natural cycles in patients undergoing ART. WHAT IS KNOWN ALREADY Letrozole is the most commonly used aromatase inhibitor for mild ovarian stimulation in ART. However, its safety in terms of pregnancy and neonatal outcomes is unclear. STUDY DESIGN SIZE, DURATION This retrospective cohort study used data from the Japanese national ART registry from 2011 to 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 3136 natural cycles and 792 letrozole-induced cycles associated with fresh, single-embryo transfer and resulting in a clinical pregnancy were included in the analysis. The main pregnancy outcomes were miscarriage, ectopic pregnancy and still birth, and the neonatal outcomes were preterm delivery, low birth weight, small/large for gestational age and major congenital anomalies. Terminated pregnancies were included in the analysis of major congenital anomalies. Odds ratios (ORs) and 95% CIs were calculated using multivariate logistic regression analysis adjusted for maternal age and calendar year. MAIN RESULTS AND THE ROLE OF CHANCE The risk of miscarriage was significantly lower in women administered letrozole (adjusted OR [aOR], 0.37, 95% CI, 0.30–0.47, P < 0.001). There was no significant difference in the overall risk of major congenital anomalies between the two groups (natural cycle 1.5% vs letrozole 1.9%, aOR, 1.24, 95% CI, 0.64–2.40, P = 0.52), and no increased risk for any specific organ system. Subgroup analysis demonstrated that the risk of major congenital anomalies was not increased in patients who underwent either in vitro fertilization or ICSI, or in those who received early cleavage stage or blastocyst embryo transfer. All other pregnancy and neonatal outcomes were comparable between the two groups. LIMITATIONS REASONS FOR CAUTION Despite the large sample size, we were only able to rule out the possibility that letrozole might cause large increases in birth-defect risks in ART patients. WIDER IMPLICATIONS OF THE FINDINGS The results suggest that letrozole stimulation reduces the risk of miscarriage, with no increase in the risk of major congenital anomalies or adverse pregnancy or neonatal outcomes compared with natural cycles in women undergoing ART. Letrozole may thus be a safe option for mild ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER Not applicable.

Localization and release of immunoreactive vasoactive intestinal polypeptide in bovine adrenal medulla

The concentration of immunoreactive (IR) vasoactive intestinal polypeptide (VIP) in extracts from bovine adrenal medulla was 29.9 +/- 7.2 pmol/g wet wt., which was about 100 times that of IR neurotensin and 30 times that of IR somatostatin. Chromatographic analysis showed that most of the IR-VIP was the same molecular size as synthetic VIP(1-28). On retrograde perfusion of isolated bovine adrenal gland, release of VIP with catecholamine (CA) was marked on stimulation with high K+, but slight on stimulation with acetylcholine, which induced marked release of CA. These results suggest that most of the VIP is localized not in CA storing granules in chromaffin cells, but in other intraadrenal neuronal components. In immunohistochemical studies, IR VIP fibers with large varicosities were observed around the vessels in the adrenal medulla.