Human epidermal growth factor (hEGF), a potent growth stimulator of many tissues in culture, has been isolated from human urine and subsequently identified in many human biological fluids including breast milk. In this study, partial purification and characterization of hEGF-like substance (s) in human milk were performed using homologous hEGF radioimmunoassay (RIA) and radioreceptor assay (RRA).
hEGF-like material (s) was extracted from pooled human milk by ethanol precipitation, followed by adsorption to cation- and anion-exchange resin. DEAE-Sephadex G-25 ion-exchange chromatography of human milk extracts revealed three major components with hEGF activity (peak I, II, III) eluted with a linear gradient by ammonium acetate. The competitive binding curves for these components were parallel to those for standard hEGF in both RIA and RRA. The apparent molecular weight of peak I was -6, 500 and that of peak II and III was -7, 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The pI value for peak I was -4.5 and that for peaks II and III was -5.0 by isoelectric focusing. These data are comparable to the size and charge heterogeneity of hEGF in human urine extracts.
In conclusion, the major components of hEGF in human milk appear to be physicochemically, immunologically and biologically (receptor binding activity) indistinguishable from hEGF of urinary origin.

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The effects of intraperitoneal and intra-third ventricular administration of morphine on the hypothalamic corticotropin-releasing factor (CRF) and the pituitary-adrenocortical activity were examined in unanesthetized, freely moving rats. Hypothalamic CRF was measured by rat CRF radioimmunoassay.
Intraperitoneal or intra-third ventricular administration of morphine increased blood concentrations of ACTH and corticosterone while intraperitoneal administration tended to increase CRF concentration in the whole hypothalamus including the median eminence and intra-third ventricular administration increased CRF concentration in the hypothalamus excluding the median eminence. However, morphine seemed to inhibit the increase in CRF concentration in the hypothalamus induced by the ether-laparotomy stress. The main site of morphine action on the hypothalamo-pituitary-adrenocortical system seemed to be in the hypothalamic area.

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The effect of the pituitary-adrenal axis on epinephrine synthesis in the human adrenal medulla was examined by the estimation of the 24-h urinary epinephrine level after treatment with glucocorticoids in four patients with systemic lupus erythematodes (SLE), one patient with rheumatoid arthritis (RA) and one patient with adrenal pheochromocytoma. 24-h urinary catecholamines (CAs) were measured by HPLC before and after glucocorticoid treatment, dexamethasone or predonisolone was orally given for more than seven days to patients with SLE, RA or isloated ACTH deficiency and five days to a patient with adrenal pheochromocytoma.
In patients with isolated ACTH deficiency, the 24-h urinary epinephrine level was significantly lower than the normal range. In patients with SLE or RA, the 24-h urinany epinephrine level was normal and it was significantly suppressed by therapeutic doses of prednisolone 30-40mg/day. In a patient with adrenal pheochromocytoma, 24-h urinary epinephrine was extremely high and it was significantly increased after dexamethasone 0.5mg/day.
These results suggest that epinephrine synthesis in the human adrenal medulla may be dependent on the pituitary-adrenal axis. But the increase in epinephrine synthesis due to dexamethasone in a patient with pheochromocytoma may reflect the direct effect via the feeding artery to the tumor, as previously shown in an in vitro culture system.

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In order to reevaluate the earlier varying data regarding circulatory gonadotropin-releasing hormone (GnRH), we assayed extracted GnRH from the plasma frequently collected at mid-cycle in 11 women. For the analysis of episodic GnRH patterns and basal levels, blood samples were obtained at 6 h intervals for 72 h and at 15min intervals for 2h every 12h throughout the experimental period.
All blood samples were assayed for GnRH and selected samples for LH, FSH, estradiol and progesterone. For GnRH assay, 5 or 6ml of blood was mixed with 60mg of ethylenediaminetetraacetic acid, disodium salt, and 3mg of phenylmethylsulfonyl floride immediately after blood collection. These enzyme inhibitors prevented the destruction of GnRH in the blood at room temperature for at least 4 h. Plasma GnRH was extracted through several steps including florisil absorption, acidic extraction and washing with organic solvent. Nonspecific immunoreactivity in the plasma was markedly decreased through this extraction process.
Our assay values (approximate range, 0.1-2.0pg/ml) of plasma GnRH in normal women corresponded to the low range of those obtained by others who used the alcohol extraction method. The basal levels of GnRH did not change significantly throughout 3 different periods, i.e., before, during and after the LH surges, and fluctuated between a small range of 0.11 and 1.44pg/ml. Although the peak levels of GnRH observed in its episodic patterns did not change between the periods before and during the LH surges, they decreased significantly after the LH surge compared with those seen during the LH surges (0.93±0.07 vs 1.17±0.09pg/ml, p<0.05).
The present data demonstrate that immunoreactive GnRH in the extracted peripheral plasma does not change significantly in its mean, basal and peak levels during the periovulatory period except for a minor but significant decrease in the peak levels shortly after an LH surge.

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To elucidate the role of prolactin on testicular function, we treated mature rats with ovine prolactin (oPRL) and investigated the dose and timedependent changes in testicular LH, FSH and prolactin receptors as well as in serum gonadotropin and steroid levels. Twelve week-old rats were injected sc with a single dose of various amounts of oPRL (0.2, 1 and 5 IU) and killed on the first, second and third days after the treatment. Testicular LH receptor decreased to 59% of the control level as a function of time while prolactin receptor increased to 244% maximally of the control level on the second day. In contrast, FSH receptor changed in a different fashion. Smaller amounts of oPRL (0.2 and 1 IU) raised the receptor level to 193% of the control level on the first day whereas a larger amount (5 IU) did not change the receptor, which tended to remain in a low level throughout the experimental period. The serum FSH level significantly increased in every group on the second day, then returned to the control range by the third day. On the other hand, the serum testosterone level changed in a characteristic manner, decreased significantly in every group on the first day though not in a dose-dependent fashion, returned to normal on the second day and significantly increased in the 0.2 IU group on the third day (p<0.01). Similarly, the serum estradiol level decreased in the oPRL-treated groups on the first day and was restored on the second day. These results suggested that exogenously administered prolactin modulated the testicular LH, FSH and prolactin receptors, which might be subsequently related to the decreases in estrogen and testosterone secretion. These findings with prolactin may be, in part, relevant to hypogonadal function in man with hyperprolactinemia. rat; testis; receptor regulation; LH, FSH and prolactin receptors; hyperprolactinemia.

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A 51-yr-old male patient with a 3yr history of Cushing's syndrome is described. The baseline plasma cortisol level was elevated, while the plasma ACTH levels remained at an undetectable level. Dynamic testing of pituitaryadrenal function revealed no suppression after 8mg of dexamethasone, and there was no response to metyrapone or CRF, while plasma cortisol showed a hyperresponse to synthetic ACTH. Plasma cortisolresponded to insulininduced hypoglycemia without an obvious ACTH response. These and the computerized tomography data suggested a “huge” bilateral nodular adrenocortical hyperplasia which was later confirmed by surgery, The left and right adrenal glands weighed 55 and 76g, respectively, In vitro experiments, using the adrenal tissue, showed that there was an adrenal cortisol response to 1-39 ACTH but not to regular insulin, arginine vasopressin, angiotensin II, norepinephrine or epinephrine. These results indicate that plasma cortisol responded to a slight hypoglycemia-induced plasma ACTH change which was not detected in the ACTH radioimmunoassay or to factors other than ACTH which might be induced by hypoglycemia.

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The influence of bovine thyroglobulin (Tg) and/or staphylococcus aureus cowan I (SAC) on Tg antibody synthesis has been studied using cultures of 8 Hashimoto's and 5 normal peripheral blood lymphocytes (PBL). The detection of Tg antibody in the culture supernatants was performed by sensitive biotinavidin solid phase enzyme immunoassay. By using this technique, we were able to detect small amounts of Tg antibody synthesized by cultured Hashimoto's PBL responsive to bovine Tg and/or SAC; PBL from three out of eight patients produced increased levels of Tg antibody in the presence of 0.02 μg/ml bovine Tg. On the other hand, PBL from two other cases among them which were unresponsive to bovine Tg alone became responsive to bovine Tg following simultaneous stimulation with SAC. PBL from the other three cases failed to respond to bovine Tg or simultaneous stimulation with bovine Tg and SAC. The former five patients had serum Tg tanned red cell hemagglutination (TGHA) titers greater than 1: 409, 600 except in one case and the latter had serum TGHA titers less than 1: 12, 800.
These results indicated the presence of the different functional stages of Bcells to produce Tg antibody in the circulation of Hashimoto's patients and suggested that sufficient number of lymphocytes responsive to bovine Tg are present in the circulation of Hashimoto's patients with high titers of serum TGHA.

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To examine the effects of acute stimulation on the peripheral and central renin-angiotensin system, simultaneous sampling of blood and cerebrospinal fluid (CSF) for measurements of plasma renin activity (PRA), plasma angiotensin I-immnunoreactivity (PAng I-ir), plasma angiotensin II-immunoreactivity (PAng II-ir), plasma angiotensinogen and cerebrospinal fluid angiotensin II-ir (CSF Ang II-ir) and CSF angiotensinogen was carried out following intravenous injection of furosemide (5mg/kg) in conscious dogs. Administration of furosemide induced marked increases in PRA, Ang I-ir, PAng II-ir and CSF Ang II-ir, however, neither plasma nor CSF angiotensinogen was changed. Furthermore, a relatively large dose (20mg/kg/min) of intravenously infused synthetic Ang II for 20min produced a five-fold increase in PAng II-ir compared with no significant increase in CSF Ang II-ir In spite of significant suppression of PRA and PAng I-ir, there were no significant changes in either plasma or CSF angiotensinogen.
These results primarily suggest that the peripheral and the brain reninangiotensi n systems may be linked and that acute changes in the peripheral renin-angiotens in system do not alter either plasma or CSF angiotensinogen.

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A distribution of immunoglobulin G (IgG) subclass of anti-thyroid peroxidase (TPO) autoantibodies was studied to know whether anti-TPO autoantibodies are closely implicated in the pathogenesis of human autoimmune thyroid diseases. As a result of analyzing 14 patients' sera, 7 with Graves' disease and 7 with Hashimoto's thyroiditis, anti-TPO autoantibodies were found to consist of mainly IgG₁ subclass. Percentages of both IgG₁ and IgG₂ subclasses in IgG class of autoantibodies corresponded to those in the normal serum composition, whereas IgG₃ subclass was scarcely contained in anti-TPO autoantibodies and IgG₄ subclass markedly increased. It was thought that anti-TPO autoantibodies had a capability to lyse thyroid follicular cells by the mechanism of antibody-dependent complement-mediated cytolysis, because IgG₁ and IgG₂ subclasses of antibodies can fix complement and TPO locates in apical membrane surface of thyroid follicular cells.
Comparing Graves' disease with Hashimoto's thyroiditis, mean percentages of both IgG₁ and IgG₂ subclasses of 2 groups were statistically different. Namely, sera of patients with Graves' disease had higher and lower mean percentages of IgG₁ and IgG₂ subclasses of autoantibodies, respectively, than those with Hashimoto's thyroiditis, though no plausible explanation for these differences can be offered at the present time.

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To determine whether GnRH modifies prolactin (PRL) secretion in response to thyrotrophin-releasing hormone (TRH) in normal women, a group of eleven normal women, 23 to 40 years of age, was studied in the mid-follicular phase of the menstrual cycle. The PRL response to TRH was evaluated in serum under control conditions and after GnRH infusion. GnRH administration augmented basal PRL release and amplified TRH-induced PRL release. These results suggest that GnRH may be involved in PRL release, partly by increasing the sensitivity of the lactotrophs to TRH.

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Responses of growth hormone (GH) release to synthetic human growth hormone-releasing factor (hGRF)-44-NH₂ analogs were determined, and the GH-releasing potency based on dose per kg of body weight (bw) was compared with that of hGRF-44-NH₂ in female dairy calves. Four-and 12-month-old calves were injected intravenously with 0.25μg of hGRF-44-NH₂ or its analogs per kg of bw. Blood samples were collected before, and during 180min after each injection, and plasma GH concentrations were measured by radioimmunoassay. Areas under the GH response curves for 180min after injection of hGRF-44-NH₂ and its analogs were used as an index of the GH-releasing potency of each peptide. The GH-releasing potency of hGRF (1-26)-NH₂ was significantly lower than that of hGRF-44-NH₂ (P<0.05). On the other hand, hGRF (1-29)-NH₂ possessed similar potency to hGRF-44-NH₂.[D-Tyr1]-hGRF-44-NH₂ showed prolonged GH-releasing activity, though its potency was similar to that of hGRF-44-NH₂. Also, [D-Ala²]-hGRF (1-29)-NH₂ exhibited prolonged GH-releasing activity, and its potency was 2.5 (P<0.05) and twice (P<0.05) as great as that of hGRF-44-NH₂ and hGRF (1-29)-NH₂, respectively. These results demonstrate that the N-terminal 29 amino acid residues of hGRF possess the activity site required for full GH release in vivo, and [D-Ala²]-hGRF (1-29)-NH₂ has longer and greater activity, on a dose basis, than hGRF-44-NH₂ in the calves.

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Homogenates of human adult liver are capable of aromatizing norethindrone (17α-ethynyl-19-nortestosterone) to ethynylestradiol (17α-ethynylestradiol). The evidence of ethynylestradiol formation was obtained using a Bio-Rad AG1-X2 column, thin layer chromatographies and co-crystallization. Neither acid nor base was used in any step in product identification.

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