The changes in the levels of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) in the neurointermediate lobe of the pituitary (NIL) following hypertonic saline administration were examined in rats. The plasma osmotic pressure in rats receiving 2% NaCl for 8 days was greatly increased. Plasma AVP concentration in rats receiving 2% NaCl for 8 days were significantly higher than in control rats (566% of the control level). Plasma corticosterone was significantly higher in the saline-treated rats than in controls, whereas plasma ACTH was not significantly different. The pituitary ACTH concentration was much higher in the saline-treated rats than in controls. CRH in the NIL was increased significantly by saline treatment (419% of the control concentration), whereas the CRH in the paraventricular nucleus and median eminence of control and saline-treated rats did not differ significantly. The AVP in the NIL fell greatly in saline treated rats. The extract from both control and saline-treated rats showed a major peak for immunoreactive CRH, with a retention time identical to that of rat CRH. However, the peak was much higher in the extract from saline-treated rats. The immunoreactive AVP peak was greatly reduced in saline-treated rats. These results suggest that hypertonic saline administration increases the CRH in the NIL and causes AVP hypersecretion and/or hyperfunction of magnocellular-NIL CRH might be responsible for pituitary-adrenal stimulation in saline-treated rats.

View full abstract

Insulin-like growth factor II (IGF-II) levels in human plasma were measured in physiological and pathological conditions by radioimmunoassay (RIA) with biosynthetic IGF-II. This RIA was specific for IGF-II and cross-reactivity with IGF-I was 1%. The sensitivity was 15pg/tube with 50% displacement at 50pg/tube. The intra- and inter-assay coefficients of variation for IGF-II were 6.3 and 9.3%, respectively.
The plasma IGF-II levels in normal adults, patients with hypopituitarism and patients with active acromegaly were 589.6±15.8, 800.9±45.6 and 330.3± 24.3ng/ml, respectively. After human growth hormone (hGH) treatment in hypopituitarism, IGF-II slightly increased, but not significantly. After adenomectomy in patients with acromegaly, IGF-II significantly decreased. These data indicate that IGF-II concentrations in plasma were partially GH dependent. This GH dependency was less than that of IGF-I. IGF-II was low in patients with anorexia nervosa and with liver cirrhosis and high in patients with renal failure. In two cases with extrapancreatic tumorassociated hypoglycemia, plasma IGF-II was increased to 1123.8 and 843.5 ng/ml, and returned to normal after tumor resection.
These data showed that IGF-II was partly dependent on GH and nutritional coditions and that IGF-II was the most likely cause of some cases of hypoglycemia with extrapancreatic tumor. This specific and sensitive RIA of IGF-II would be useful in evaluating its physiological and pathological role in plasma and tissue.

View full abstract

Early prenatal diagnosis of 21-hydroxylase (21-OHase) deficiency would enable treatment to be done to protect the fetus from masculinization and/or life-threatening adrenal crisis at birth. We report here the prenatal diagnosis of 21-OHase deficiency with human complement component C4 cDNA to probe DNA from chorionic villi at 10 weeks of gestation. Southern analysis with human C4 cDNA identified TaqI restriction fragment length polymorphisms (RFLPs) in the family. Family analysis with these RELPs showed that the fetus was not affected at greater than 99% probability, because the frequency of recombination between the 21-OHase B gene and the C4 gene would be extremely low.

View full abstract

Using a functioning rat thyroid cell line (FRTL-5), we examined the effects of some cytokines, particularly interleukin- l (IL-1) on the growth of thyroid cells. In 5H medium, namely Coon's modified Ham's F-12 medium supplemented with 5% calf serum and a five-hormone preparation consisting of insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lysine acetate and somatostatin, IL-1 enhanced the growth of FRTL-5 cells detected by [³H] TdR incorporation. However, in 6H medium (5H medium plus bovine TSH), IL-1 inhibited the growth of FRTL-5 cells. Both effects were neutralized by the addition of anti-IL-1 antibody. Furthermore, IL-1 inhibited the growth of FRTL-5 cells induced by forskolin which is known as an adenylate cyclase activator. FRTL-5 cells have specific IL-1 receptors detected by the binding of ¹²⁵Ilabeled IL-1α. By Scatchard plot analysis, the numbers and the dissociation constants of IL-1 receptors on FRTL-5 cells were shown to be 5225/cell and 8.69×10^-10 M. Interleukin-2, interleukin-6 and interferon-γ(IFN-γ) had no significant effects on the cell growth in 6H medium, while IFN-γ and insulinlike growth factor I stimulated cell growth somewhat in 5H medium. These results suggest that IL-1 plays a regulatory role in the growth of thyroid cells through binding to the IL-1 receptors.

View full abstract

Plasma growth hormone (GH) levels during oral glucose tolerance test (OGTT) and during the daytime (0900-1700 h) and urinary GH excretion were determined in 6 normal subjects and 10 patients with postoperative acromegaly by means of a highly sensitive enzyme immunoassay (EIA). Acromegalic patients were divided into two groups according to their GH responses during OGTT. In group I patients plasma GH was suppressed below 1.0μg/l as in normal subjects, whereas GH levels persisted above 1.0μg/l in group II patients. Plasma GH profiles during the daytime were characterized by low nadir GH levels in normal subjects 0.21± 0.10μg/l and group I patients (0.38±0.10μg/l) when compared with those in group II patients (4.7±2.0μg/l). Nadir GH levels during the daytime coincided with those during OGTT. Urinary GH excretions in group I patients (median 12ng/day, range 4.3-29ng/day) were within normal limits (2.6-20ng/day) and significantly lower than those in group II patients (median 40ng/day, range 19-130ng/day.) Determination of plasma GH levels during OGTT and urinary GH excretion by the highly sensitive enzyme immunoassay is useful in evaluating the activity of postoperative acromegaly.

View full abstract

To ascertain the presence of thyroid growth-promoting activity (TGA) in the sera of pregnant women, we measured TGA in the sera of pregnant women by means of a bioassay based on [³H]-thymidine ([³H] Tdr) incorporation in cultured rat FRTL-5 thyroid cells. Furthermore, to elucidate the mechanisms of human chorionic gonadotropin (hCG) in promoting the thyroi growth, we evaluated the effects of blocking type TSH receptor antibody (blocking IgGs) from patients with primary hypothyroidism on the activity of hCG. After the PEG-pretreated serum or the serum plus blocking IgGs wasincubated for 72h at 37α with FRTL-5 cells and [³H] Tdr, [³H] Tdr incorporated in the cells was counted. Although 9 normal pregnant women had normal TGA, two patients with hydatidiform mole, whose hCG levels were 966, 500 and 497, 100 IU/L, had positive TGA, but the activity showed normal when analyzed with the addition of a blocking IgG. hCG also showed a dosedependent increase in [³H] Tdr incorporation, and it was inhibited by the addition of blocking IgGs. Furthermore, the inhibition of hCG-induced [³H] Tdr incorporation by 16 blocking IgGs correlated with their TBII and the inhibition activity of hCG-induced cAMP accumulation. Analysis by the Lineweaver-Burk plots of dose response curves of TSH- and hCG-induced [³H] Tdr incorporation showed the same inhibition pattern as with the addition of the same blocking IgGs. In conclusion, 1) hCG-related TGA exists in the sera of some patients with hydatidiform mole; and 2) hCG and the sera of some patients with hydatidiform mole promote thyroid growth, at least in a part, via TSH-receptors in FRTL-5 cells.

View full abstract

On days 5, 10, 15 and 20 of pregnancy, rat corpora lutea (CL) were dissected and dissociated into single cell suspensions by enzyme treatments. The suspended luteal cells were allowed to sediment in a BSA gradient at 4°C for 3.5 hours. Five fractions were collected from the top (Fraction (Fr.) 1) to the bottom (Fr. 5) of the gradient. Cells were incubated in serum-free DME-F12 for 20 hours with or without hCG (100 ng/ml) to test them functionally, and the accumulation of progesterone and testosterone was determined by radioimmunoassay. To assess 3β-hydroxysteroid dehydrogenase (HSD) activity, a histochemical suspension-staining procedure was used. Cells were examined by light microscopy, and the percentage of cells containing dark blue formazan deposits and their diameters were determined in at least 40 microscopic fields.
The number of cells staining for 3β-HSD did not vary by day 15 but decreased from 141.6±16.5×10³ cells/CL on day 15 to 113.8±13.2×10³ cells/CL on day 20 of pregnancy. However, 3β-HSD-positive cells maintained the same levels of progesterone secretion until the advent of luteolysis, then they increased in size progressively throughout pregnancy. In BSA gradients, the relatively larger 3β-HSD-positive cells migrated faster than the smaller 3β-H SD-positive cells on each day of pregnancy. The diameters of 3β-HSDpositive cells differed significantly in Frs. 2, 3, 4 and 5 on days 15 and 20 of pregnancy. On day 15 of pregnancy, less progesterone accumulated in wells containing 3β-HSD-positive cells from Fr. 2 (mean diameter; 24.96 μm) than from Fr.3, Fr.4 and Fr.5 (mean diameters; 27.20, 30.79 and 31.28 pan, respectively) but the Fr. 2 cells responded more to hCG stimulation. Fr. 2 also showed a higher ratio of testosterone accumulation to progesterone accumulation than the other fractions. The response to hCG stimulation of cells in Fr. 2 tended to be higher than that in Fr. 3 on day 20 of pregnancy. These data suggest that the steroidogenic rat luteal cells are comprised of morphologically and functionally different cell types after day 15 of pregnancy. No stimulating nor inhibiting effects were observed in co-incubations of cells from Fr. 2 with cells from Fr. 3 or Fr. 4 on days 15 and 20 of pregnancy.

View full abstract

In order to clarify the effect of thyroid hormone on the plasma atrial natriuretic peptide (ANP) concentration, 14 patients with Graves' disease and 6 normal control subjects were studied. They were all under constant sodium intake because dietary sodium is known to affect the amount of plasma ANP. Sodium intake remained constant at 171 mEq daily for five consecutive days at which time the ANP concentration was measured. Graves' disease patients were tested both before and after surgery. The preoperative, hyperthyroid ANP level concentration in Graves' disease patients was 6.7±2.3 fmol/ml compared to a significantly lower level of 4.2±1.4 fmol/in normal control subjects. Seven days after surgery when Graves' disease patients became euthyroid their ANP markedly decreased to 4.2±2.9 fmol/ml.
In the present study we were able to confirm that under a constant sodium diet, high plasma ANP in patients with Graves' disease was reduced after surgery when they became euthyroid. Results also suggest that high circulating ANP might play an important role in sodium and water metabolism and hemodynamic changes in hyperthyroidism.

View full abstract

To clarify the indication of surgery in incidentally discovered asymptomatic adrenal masses, we analyzed 386 Japanese cases, 379 cases reported in Japan during the past 25 years (from 1964 to 1988) and 7 cases from our own experience. From a total of 460 patients, we carefully selected 379 patients satisfying our criterion of the absence of symptoms and signs suggestive of active hormone over-secretion as described in each case report. From the Japanese series, there was a high incidence of pheochromocytoma patients (20 of 37 patients) who had no symptoms and signs but had high plasma or urine catecholamines. Scintigraphy with ¹³¹I-meta-iodo-benzyl-guanidine was useful in the diagnosis of pheochromocytoma. For the other asymptomatic adrenal tumors, except for myelolipoma and adrenal cyst, differential diagnosis between malignant and benign adrenal lesions by imaging procedures such as whole body computed tomography (CT), ultrasonography (US), adrenocortical scintigraphy, and angiography was not always possible. In addition, among the 109 patients with cortical tumors whose hormonal data were reported, no clear-cut differentiation of malignant tumor from benign by means of these data could be obtained. Since 1980 whole body CT scanner and high resolution US scanner have become widely available, and there have been 283 cases of asymptomatic adrenal tumors who satisfied our criterion. Cortical carcinomas smaller than 3 cm and 6 cm in diameter account for 3.8% and 6.6%, respectively, of the total of 101 cases of cortical carcinoma, cortical adenoma, ganglioneuroma, and hemangioma during this period. The size of the smallest cortical carcinoma with metastasis was 2 cm in diameter in this series. Pre-operatively, an adrenocortical carcinoma 2.8 cm in diameter in our patient could not be diagnosed as such by imaging techniques and measurement of plasma hormones. These findings suggest that an adrenal mass larger than 3 cm should be removed and a patient with a smaller cortical tumor should be carefully followed up.

View full abstract

The paradoxical effects of TAP-144-SR, a biodegradable sustained-release formulation of a potent GnRH agonist (TAP-144, leuprolide acetate) were evaluated in male rats by comparing its potency with that of TAP-144 solution.
A single sc injection of TAP-144-SR (equivalent to 0.1mg/kg/day as TAP-144), prepared by encapsulating the agonist in microcapsules of copoly (DLlactic/glycolic acid), suppressed serum levels of androgens, and the levels remained suppressed for 4 weeks.
The potency of the paradoxical effects of TAP-144-SR was evaluated 4 weeks after treatment by comparing it with that of TAP-144 solution administered daily for 4 weeks. Both daily injections of TAP-144 solution and a single injection of TAP-144-SR (equivalent to 0.02, 0.2 or 2mg/kg/day as TAP-144) decreased the weight of the testes, prostates and seminal vesicles in a dose-dependent manner in a 4-week assay in male rats. TAP-144-SR was more effective than TAP-144 solution in reducing these organ weights. Serum and pituitary concentrations of LH and FSH and serum testosterone levels were also lower in TAP-144-SR-treated than in TAP-144 solution-treated rats. These results indicate that the paradoxical effects were more extensive upon TAP-144-SR treatment, suggesting that maintaining constant serum TAP-144 levels results in more extensive desensitization of the pituitary and testes. These results also suggest advantages of TAP-144-SR over TAP-144 solution in both efficacy and convenience as an anti-prostatic tumor agent.

View full abstract

In order to determine the changes in the expression of LHβ messenger ribonucleic acid (mRNA) during GnRH agonist (GnRHa) treatment (0.94mg/28 days), the concentration of the mRNA of LHβ was assessed together with the serum LH concentration, pituitary LH content and LH response to GnRH at various times during long-acting GnRHa treatment in immature female rats.
The serum LH concentration was increased at hour 1, gradually decreased starting at approximately hour 3 and had returned to the control level on day 28. Pituitary LH began to decrease at hour 3. The concentrations of LHβ mRNA were not significantly different from those in the control group from hour 1 to hour 18, but were lower from day 3 to day 28. Serum LH response to native GnRH (1μg) began to be inhibited on day 7.
These results indicate that the short term treatment with GnRHa stimulates the release of preformed LH rather than synthesis of LHβ mRNA and that the long term treatment inhibited the expression of LHβ mRNA in a time dependent manner.

View full abstract

Obesity is usually associated with expansion of blood volume. Therefore, we studied whether obesity affects cardiac and plasma atrial natriuretic peptide (ANP) levels in experimental animal model. Mice made obese with gold thioglucose developed cardiac hypertrophy associated with increases in ANP in atrial tissue and plasma. There were significant (p<0.01) correlations between the cardiac ANP concentration and body weight or cardiac weight. These data suggest that enhanced synthesis of atrial ANP in obese mice can be mainly ascribed to increased blood volume associated with cardiac hypertrophy.

View full abstract

The effects of chemical diabetes and fasting on fuel metabolism and insulin secretory activity in late pregnancy were investigated. Female Wistar rats were made chemically diabetic (CD) by intravenous injection of streptozotocine (30 mg/kg) 2 weeks before conception. When CD pregnant rats were fed, plasma glucose and insulin levels were not significantly different from those of normal pregnant rats. Ketone body levels, however, were higher in CD pregnant rats than in normal pregnant rats, indicating insulin resistance in CD rats. Insulin secretion from the perfused pancreas caused by arginine or glucose was markedly decreased in CD pregnant rats. The pregnant rats were fasted for 2 days, from day 19 to 21 of gestation. Plasma glucose and insulin concentrations decreased similarly in the two groups, whereas ketone body concentrations in CD pregnant rats were significantly higher than those in normal pregnant rats. Glucose-induced insulin secretion by the perfused pancreas was markedly attenuated by fasting and was not significantly different in normal and CD pregnant rats. These observations suggest that diabetes mellitus accelerates starvation in late gestation, due to increased insulin resistance and poor insulin secretion, and that fasting in diabetic pregnancy amplifies ketogenesis.

View full abstract

The therapeutic effect of sustained-release microspheres of a potent LHRH agonist (leuprorelin acetate) on experimental endometriosis in female rats was examined histologically. Endometriosis was produced in rats by autotransplantation of endometrial tissue obtained from the left uterine horn into the renal subcapsular space.
In the nontreated rats, the transplants were well established and had formed large cysts containing fluid. The walls of the cysts were composed of epithelium and stroma resembling that of normal endometrium. In the rats which received the microspheres of leuprorelin acetate, growth of the transplant was markedly suppressed as evidenced by the reduced size of the cystic cavity and the flattened and pyknotic epithelium. Also, the uterine and ovarian weight decreased significantly. In the ovariectomized rats, growth of the transplant was also markedly suppressed, and the uterine weight decreased.
The present results clearly indicate that a single injection of the sustainedrelease microspheres of leuprorelin acetate markedly suppresses growth of the transplant and produces uterine and ovarian atrophy in the rats.

View full abstract

Placental sulfatase deficiency (PSD) is a rare disorder with low estrogen production due to placental enzymatic deficiency. Although many papers have been published on this enzymatic deficiency, little information is available on steroidal environment in newborn babies from PSD mothers. Seven cases of PSD were confirmed and serum concentrations of nine steroids which included cortisol, free and conjugated pregnenolone, 16α-hydroxypregnenolone, DHA and 16α-hydroxy-DHA in cord blood at delivery and peripheral veins during the neonatal period were measured by radioimmunoassay. In all seven instances, healthy male infants were delivered, but six of the babies developed ichthyosis.
Conjugated Δ⁵-steroid concentrations in cord blood were found to be high, while 16α-hydroxypregnenolone was low when the PSD cases were compared with the controls. In the PSD cases, free 16α-hydroxy-DHA and 16α-hydroxypregnenolone were both lower than in controls during the first seven days after birth. These results indicate that the production of Δ⁵-C₂₁ steroid in PSD infants is limited.
In the present study of newborn infants with PSD, the pattern of circulating steroids was first demonstrated and the relationship between the sulfatase activity in the neonates and ichthyosis was discussed.

View full abstract

A case of anaplastic, multinucleated giant cell tumor of the thyroid was studied by light and electron microscopy. The coexistence of anaplastic sarcomatous tumor and well differentiated follicular carcinoma, and the presence of desmosomes among the mononuclear cells suggested that this tumor originates in thyroid follicular cells. The multinucleated giant cells, which characterize this thyroid tumor, appeared to be formed by fusion of follicular carcinoma cells and mononuclear epithelial cells, and not by nuclear division without cytoplasmic division.

View full abstract

The serum concentration of hyaluronan (HA) was measured by radiometric assay in patients with pretibial myxedema (PTM) and Graves' ophthalmopathy (GO). The mean HA concentration in the patients (n=8) was 21.2±15.3 (mean±SD)μg11, while that of Graves' disease without skin or eye involvement (n=7) was 23.5±11.0 (mean±SD)μg/l and that of the control (n=8) was 25.5±16.4 (mean±SD)μg/l.
We conclude that local accumulation of glycosaminoglycan in PTM or GO is not associated with an increase in the serum HA concentration.

View full abstract

A 34-year-old man had a huge hormone-producing adrenal cancer with multiple lung metastases, direct liver invasion and a tumor thrombus in the inferior vena cava. Initial treatment was mitotane alone. The dose of mitotane was 2g/day initially and gradually increased to 15g/day in combination with 600mg of tegafur per day. During the initial phase of chemotherapy, the serum mitotane level was relatively low (2.9-4.6μg/ml) and the pulmonary metastases tended to grow in size in spite of a gradual decline in urinary 17-KS and 17-OHCS and a regression of the primary tumor. A seemingly marked increase in serum mitotane (20.5-34.5μg/ml) was coincident with the addition of tegafur. Rapid and consistent regression of the primary tumor occurred. This excellent response to the chemotherapy made the primary tumor with liver invasion and the metastases resectable. The adverse effect of mitotane, central nervous toxicity, appeared to be serum mitotane level dependent. The present results, together with previous reports in the literature, seem to recommend the following therapeutic approaches to advanced adrenal cancer: monitoring of the serum level may be useful in predicting the efficacy as well as the occurrence of side effects of mitotane, surgical treatment of the lesions should be performed whenever possible, even though it may be only palliative, and the combination of mitotane and tegafur is a choice of chemotherapy which should be evaluated in future studies.

View full abstract

Two young girls with hirsutism and premature pubarche showed nonclassical 3β-hydroxysteroid dehydrogenase (3β-HSD) deficiency. Post-ACTH increased serum Δ⁵-17-hydroxypregnenolone and increased ratio of Δ⁵-17-hydroxypregnenolone/17-hydroxyprogesterone are the most sensitive indicators of nonclassical 3β-HSD deficiency. Nonclassical 3β-HSD deficiency may not be uncommon, but most cases may have gone unrecognized. Routine assay of Δ⁵-17-hydroxypregnenolone should be made generally available.

View full abstract