We present here the results of water restriction test (WRT) and hypertonic saline loading test (HSLT) in normal children. Maximal urine osmolality during WRT (W-Umax; 1040±154mOsm/kg) may be age-dependent (W-Umax=812+23age, r=0.52, p<0.05), although maximal arginine vasopressin (AVP) levels during WRT did not show any correlation with age. The relationship between plasma osmolality (Posm) and AVP during HSLT in children (AVP=0.31(Posm-277)) was similar to that in normal adults. A plateau urine osmolality during HSLT (H-Umax) was 713±109mOsm/kg. It did not increase with age. AVP levels 3 h after the infusion did not correlate with age. Minimal AVP and Posm values (about 6pg/ml, 295mOsm/kg, respectively) for creating H-Umax apparently existed during HSLT. The minimal AVP value (about 6pg/ml) for H-Umax (during HSLT) was higher than the AVP levels (2.41±1.37pg/ml) at W-Umax (during WRT). W-Umax (1040±154mOsm/kg) was significantly higher than H-Umax (713±109 mOsm/kg). Judging from the above comparison of AVP and Uosm (W, H-Umax) at the plateau state of WRT and HSLT in normal children, a change in the apparent tubular response to AVP may be one of the important factors to maintain circulatory volume (CV).

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One hundred and thirty-two consecutive patients with primary hyperparathyroidism were studied preoperatively for the presence of chondrocalcinosis, the roentgenographic marker of calcium pyrophosphate dihydrate (CPPD) crystal deposition disease, by obtaining radiographs of knees, wrists and pelvis. Chondrocalcinosis was found in 8 patients (6.1%), each of whom was over 50 years of age. In 72 of the patients over 50 years of age, the prevalence of chondrocalcinosis in the hyperparathyroid patients (11.1%) was greater than that found in 72 control patients (2.8%) with thyroid nodular disease who were exactly matched for age and sex, but the difference was not signicant. The prevalence of chondrocalcinosis in the hyperparathyroid patients sharply increased with age. In the group in their 50's it was 4.4%, rising to 15.8% in patients in their 60's and reaching 37.5% for those over 70 years of age. Patients with chondrocalcinosis were significantly older than those without this finding (p<0.005). Those with chondrocalcinosis also had significantly higher preoperative serum calcium levels than those without it (p<0.05). While chondrocalcinosis was detected by taking joint radiographs in all patients with primary hyperparathyroidism, acute arthritis (pseudogout attack) occurred in only 2 of the 132 patients (1.5%) after parathyroidectomy, but this represents 25%(2 of 8) of those who had chondrocalcinosis. An attack of pseudogout may therefore be one of the most common postoperative complications of parathyroid surgery in the elderly. Considering the low incidence of pseudogout attack following parathyroidectomy, preoperative radiological studies of the knee joints are sufficient to screen for chondrocalcinosis and are recommended for patients over 60 years old in Japan. Since 25% of patients with chondrocalcinosis will be expected to get actute pseudogout postoperatively, calcium supplement therapy in the immediate postoperative period is recommended for patients with this radiologic finding in order to prevent such an attack.

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We have seen a case of “diabetic non-ketotic hyperosmolar coma” with ketosis. An 84-year-old man was brought into the hospital in a deeply comatous and dehydrated state. The initial blood glucose level was 1252mg/dl with plasma osmolarity of 435 mOsm/l, but no ketonuria was detected by the nitroprusside method (Ketostix). However, the plasma 3-hydroxybutyrate (3-OHBA) level was 5 mM in a newly developed bedside film test. The serum ketone bodies were later found to be 5.56 and 0.82 mmol/l for 3-OHBA and acetoacetate (AcAc), respectively. A marked increase in glucagon, cortisol and ADH with renal dysfunction (creatinine 5.0 mg/dl) were noted. An abnormal electrocardiogram, occular convergence and chorea like movement disappeared after correction of metabolic disturbances. The moderate level of IRI (14 μU/ml) on admission and a good response to glucagon 2 months after admission also indicate that the present case is a typical hyperosmolar non-ketotic coma. Because of a preferential increase in 3-OHBA, ketonuria seemed to be absent in the regular nitroprusside test. Marked dehydration is thought to cause renal dysfunction, and the increase in ADH may have helped to prevent further aggravation of ketoacidosis. We propose to change the term hyperosmolar non-ketotic coma (HNC) to diabetic hyperosmolar coma (DHC), because sometimes patients with hyperosmolar non-ketotic diabetic coma are ketotic, as seen in the present case. Determination of 3-OHBA or individual ketone bodies in blood is important and essential for the differential diagnosis of diabetic coma. The diagnosis of either ketoacidotic or hyperosmolar coma should be made depending on the major expression of ketoacidosis or hyperglycemic hyperosmolarity.

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Differences from normal in microsomal antigen (M-Ag) may be involved in the development of autoimmune thyroid disease. We compared the M-Ag in Graves' thyroid immunologically and biochemically to that in normal thyroid. The concentration of M-Ag, measured with an enzyme-linked immunosorbent assay, was significantly greater in the Graves' microsomes than in normal microsomes. Binding of a patient's microsomal antibody to Graves' microsomes was completely inhibited when the serum was first incubated with normal thyroid microsomes. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blotting were done with a monoclonal antibody to denatured M-Ag. In both Graves' and normal thyroids, M-Ag existed as 107-, 101-, and 95-kDa peptides. After incubation with V8 protease, the residual antigenic peptide had a molecular weight of less than 60, 000 and after incubation with trypsin, 95- and 87-kDa peptides and several smaller antigenic peptides were found. There were no significant differences in the pattern of normal and Graves' microsomes after digestion. Twodimensional gel electrophoresis of Graves' microsomes showed that the isoelectric point for the 107-kDa peptide was at pH 7.2; that for the 101-kDa peptide was at pH 6.2, and that for the 95-kDa peptide was at 6.5. These values were not different from those observed for normal microsomes. These results indicate that M-Ag in Graves' thyroid does not differ from that in normal thyroid, and that microsomal antibodies in autoimmune thyroid disease probably do not arise from differences in the antigen.

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Serum Bone Gla Protein (BGP) levels were measured by both immunoradiometric assay (IRMA) and radioimmunoassay (RIA) to investigate the effect of intermittent 1, 25 (OH) ₂D3 administration to dialysis patients who could not tolerate an increase in an active vitamin D₃ dose and/or calcium to control secondary hyperparathyroidism due to hypercalcemia. The administration of active vitamin D₃ gradually increased the serum BGP to more than 3 times the original level by the 8th week. At the 12th week after starting the active vitamin D₃ therapy, mean BGP was about twice the original level, which was about half the maximum level at the 8th week. The BGP (IRMA)/BGP (RIA) ratio was increased significantly at 4th and 8th weeks compared to the original level. During this period, serum calcium, phosphorous, or intact molecule PTH (I-PTH) levels showed jnsignificant changes, with a slight reduction in the mid molecule PTH (m-PTH) level, and a significant reduction in ALP. Serum BUN and creatinine levels were not changed significantly. These data suggest that BGP was increased through direct stimulation of osteoblasts by the active vitamin D₃, and the increase was not due to deterioration of secondary hyperparathyroidism. The reduction of the increase in the BGP level at the 12th week with insignificant biochemical changes suggests that activation of osteoblasts by vitamin D₃ may be transient. In conclusion, intermittent active vitamin D₃ increases serum BGP, without deterioration of major biochemical changes even in patients with moderate to severe secondary hyperparathyroidism, although the increase may be transient. These facts suggest that the serum BGP of hemodialysis patients is controlled at least in part by active vitamin D₃. Whether the increase leads to histological amelioration of renal osteodystrophy or not remains to be determined.

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Effects of epidermal growth factor (EGF) on the development of mouse 2-cell embryos cultured in vitro were investigated. The addition of EGF at a concentration of 0.5 ng/ml enhanced the development of 2-cell embryos during 24 h of incubation. As expected, EGF stimulated the synthesis of DNA in the 2-cell embryos about 4-fold over the control. The growth-promoting effect of EGF seemed to be specific in that other growth factors, such as transforming growth factor-α(TGF-α), transforming growth factor-β(TGF-β), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), nerve growth factor (NGF) and fibroblast growth factor (FGF) had no effect on the embryonal development. The addition of EGF also increased the rate of RNA synthesis in a dose-related manner between 0.1 and 50 ng/ml. However, protein synthesis was unaffected by EGF. These results raise the possibility that EGF may participate in the process of early embryogenesis in vivo.

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Changes in interstitial collagenase activity in the rat uterine cervix during ripening were clarified in a time-dependent manner. Premature delivery was induced by an antiprogesterone agent, RU486, for rats in late pregnancy. The presence of interstitial collagenase in the extract from the rat cervical tissue was demonstrated, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using the natural and unaffected collagen as a substrate. The collagenase activity was determined as the release of digested peptides from the radio-labeled collagen. Our experiments with RU486 were performed in rats on the 18th day of pregnancy. A single administration of RU486 (15 mg/kg) resulted in the premature delivery of all treated rats within 30 h after the injection (average time was 23.9 h). The marked increase in cervical wet weight was observed up to the time to premature delivery along with a significant acceleration from 18 h after the adminsitration of RU486. In this state, the cervical collagenase activity was enhanced, the highest levels being recorded at 21 h after the administration. The interstitial collagenase in the uterine cervix appears to play a significant role in the regulation mechanisms of cervical ripening in late pregnant rats.

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Synthetic α-human atrial natriuretic peptide (hANP) was infused continuously at a rate of 80 ng/kg/min for 20 min into normal volunteers and patients with chronic renal failure (CRF) receiving hemodialysis. Blood pressure (BP) decreased significantly both in normals and in patients with CRF. The magnitude and the duration of the decrease, however, were greater in patients with CRF. The plasma aldosterone concentration (PAC) decreased significantly in normals and only minimally in patients with CRF. The half time (T1/2) of plasma hANP in patients with CRF (M±SE: 4.5±0.5 min) was longer than that in normals (1.8±0.2 min). Moreover, the metabolic clearance rate in patients with CRF (64±7ml/kg/min) was less than in normals (150±20ml/kg/min). Thus, the T1/2 in plasma of hANP in patients with CRF was noticeably longer than in a normal control group. These findings suggest that hANP suppresses PAC regardless of electrocyte imbalances and/or volume change induced by kidney dysfunction and that the kidney may be important in degrading hANP.

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The role of arginine vasopressin (AVP) in blood pressure regulation in humans and animals is still controversial. The present study was designed to investigate the effects of AVP on blood pressure and the excretion of sodium and prostaglandin (PG) E₂ in rabbits. AVP dissolved in 0.01 M acetic acid was infused subcutaneously at a rate of 0.86 ng/kg/min with a miniosmotic pump into 12 New Zealand white rabbits (2.7±3.4 kg), while 10 controls were given vehicle alone. AVP infusion resulted in a 3.5-fold rise in the level of plasma AVP (21.8±4.4 (SEM) pg/ml) as compared with controls, associated with a signifcant decrease in the urine volume and urinary excretion of sodium. The PGE₂ excretion was increased 1.8-fold after AVP infusion. In the chronic AVP-infused group, blood pressure was not significantly increased, but the acute vascular response to AVP was significantly attenuated without any changes in the vasopressor response to angiotensin II. Preadministration of V₁-antagonist completely abolished the vasopressor action of AVP, but not that of angiotensin II, in either group. These results suggest that circulating AVP within physiological range of concentrations may stimulate renal PGE₂ synthesis and attenuate the vascular response through vascular V₁ receptors without affecting the baroreflex, which may be attenuated through V₂ receptors.

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Changes in the expression rate of epidermal growth factor receptor (EGFR) mRNA with age in the liver of rats were examined to assess the contribution of EGF to the proliferation of hepatocytes in younger animals. Northern blot analyses with a ³²P-labeled probe derived from the extracellular domain of EGFR cDNA were evaluated quantitatively by normalizing the densitometric scanning data with the relative amounts of poly (A) ⁺ RNA in the samples. The expression of EGFR mRNA was low during the early period and increased towards the adult level after 21 days of birth. In contrast, the rate of β-actin mRNA expression was significantly higher immediately after birth and showed a tendency to decrease with time.

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To clarify the possible role of prolactin in the regulation of immune responses in man, we measured circulating anti-thyroid antibodies in 172 normal subjects, 84 patients with prolactinoma and 63 patients with acromegaly with normal thyroid and adrenocortical functions. Frequencies of positive thyroidal microsome and thyroglobulin antibodies were significantly (p<0.05 and p<0.01, respectively) higher in women with prolactinoma (20.6% and 20.6%) than in normal women (7.5% and 4.7%). Men with prolactinoma had a significantly (p<0.05) higher frequency of positive thyroglobulin antibody (18.8%) than normal men (1.5%). When the subjects were divided by decade, women with prolactinoma in the 4th decade had significantly (p<0.05) higher frequencies of positive thyroidal microsome and thyroglobulin antibodies (30.8% and 30.8%) than normal women of corresponding age (3.7% and 3.7%). In contrast, there was no significant difference in the frequencies of positive anti-thyroid antibodies in patients with acromegaly and in normal subjects. Analysis of the peripheral lymphocyte population revealed that patients with prolactinoma had a higher percentage of B cells than normal subjects, while there was no significant difference in the percentages of total T lymphocytes or in the helper and suppressor T cell ratios in the two groups of subjects. These results suggest that prolactin regulates humoral immune responses in man directly by stimulating B lymphocytes or indirectly by inhibiting suppressor T lymphocyte activity.

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To determine whether environmental factors could affect the incidence of diabetes in RT6.1 ⁺ lymphocytes-depleted diabetes resistant (DR) BB rats, we tested polyinosinic-polycytidylic acid (Poly I: C), as an immune activator, in conjunction with anti-RT6.1 antibody in DR-BB rats which were bred in a specific pathogen free (SPF) condition. Diabetes was induced by the combined administration of poly I: C and anti-RT6.1 antibody. The use of poly I: C or anti-RT6.1 antibody alone did not cause diabetes. These results suggest that RT6.1 ⁺ T lymphocytes regulate autoimmune diabetes and that non-specific immune activation caused by environmental factors plays a key role in inducing diabetes in DR-BB rats.

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The effect of a prolonged release formulation of recombinant DNA derived bovine somatotropin (rbST) on the plasma concentrations of growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, glucose, blood urea nitrogen (BUN) and nonesterified fatty acids (NEFA), and milk production in lactating dairy cows was studied. Eight cows were divided into two equal groups. One group was the noninjected control, and cows in the other group received a single subcutaneous injection of 640 mg rbST. Plasma GH levels in the rbST-treated cows were higher than in the control cows for 10 days after the injection. Plasma IGF-I concentrations were significantly higher in the rbST-treated cows than in the control for 14 days after the treatment. In the rbST-treated cows, the plasma concentrations of insulin and glucose tended to be higher than those in the control until 7 days after the injection. Also, plasma NEFA levels were higher in the rbST-treated cows for 10 days. In contrast, plasma BUN levels were significantly lower in the rbST-treated cows for 17 days after the treatment. For 28 days after the injection, the mean daily milk yield in rbST-treated cows was 4.5 kg (21.2%) more than that in the control cows. In the rbST-treated cows, a highly positive correlation was observed between the mean daily milk yield and the mean plasma concentration of IGF-I throughout the postinjection period. These results indicate that the rbST treatment induces the increase in the circulating concentrations of GH, IGF-I, insulin, glucose and NEFA and the decrease in BUN, and suggests that a part of the galactopoietic effect of rbST may be mediated by IGF-I.

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Two forms of placental lactogen (PL), designated PL-I and PL-II, have been reported in the serum of pregnant rats; PL-I was secreted during mid-pregnancy and PL-II was secreted near term. In the present study, we found that two distinct forms with PL-like activity were secreted into the blood during mid-pregnancy. Serum from day-12 pregnant rats was subjected to high-performance liquid chromatography (HPLC) gel-filtration (TSK-G3000SswXL column), and PL activity was assayed by radioreceptor assay using ¹²⁵I-labeled rat PRL and hepatocyte plasma membrane from pregnant rats. HPLC-gel filtration separated the PL activity into two peaks with mol wt of 55-60K and 45-50K, as estimated by reference to the elution volume of standard proteins. We tentatively designated these peaks PL-α and PL-β, respectively. Considering these mol wt, PL-β seemed to be identical to PL-I. mRNA was extracted from samples of placenta obtained each day from day 8 to day 12 of pregnancy and analyzed by means of a translation system involving micro-injection into Xenopus oocytes. The time of appearance of the mRNAs corresponding to PL-ct and PL-β did not correspond and differed according to the day of pregnancy, suggesting that there are individual mRNAs for each PL in rat placenta. Treatment of PL-α and PL-β with peptide: N-glycosidase F completely abolished the binding activity to their receptors. Also since they were sensitive to glycosidases such as endoglycosidase H, endoglycosidase F and neuraminidase, these PLs were considered to possess N-linked glycoresidue (s) and to be sialylated. Based on the results, there are two distinct PLs, which are secreted at mid-pregnancy, are glycosylated and have different mRNAs.

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The serum of rats at mid-pregnancy contains at least 2 distinct placental lactogen (PL)-like substances tentatively termed placental lactogen-α(PL-α) and placental lactogen-β(PL-β)(Endocrinol Japon 38: 533-540, 1991). We have investigated the secretory patterns of three placental lactogens (PL-α, PL-β and placental lactogen-II) during normal pregnancy and in two prolonged-pregnancy models. Pregnancy was prolonged by the introduction of new corpora lutea by inducing ovulation on day 15 of pregnancy by successive treatments with PMSG (30 IU/rat, sc on day 12) and hCG (10 IU/rat, iv on day 14), and in the second model by progesterone implants on day 15 of pregnancy. During normal pregnancy, each of the 3 PLs exhibited only one secretory peak in the serum; PL-α and PL-β on day 12 and placental lactogen II (PL-II) on day 20. Interestingly, in the rats with new sets of corpora lutea, serum PL-α and PL-β levels began to increase again on day 18 and showed peaks on day 20 for PL-β and on day 22 for PL-β. In this model, the initiation of PL-II secretion was not affected, but high levels were maintained until day 26, when parturition occurred. In rats receiving either PMSG or hCG, the secretory patterns of the PLs were similar to as those during normal pregnancy. In the progesterone-implanted rats, the second secretory peaks of PL-α appeared on day 20 and PL-β on day 22. The start of secretion of PL-II was delayed for 4 days. In the progesterone-implanted rats, ovariectomy on day 15 abolished the secretions of all 3 placental lactogens (PL-α, PL-β and PL-II). Thus, mid-pregnancy-specific PLs, PL-α and PL-β, are secreted again if the period of pregnancy is extended either by the induction of a new set of corpora lutea or by progesterone implantation, and these secretions depend on the ovary.

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Glucose-stimulated insulin release from rat pancreas is known to be blunted by aging. In the present study, we examined the effect of aging on insulin release induced by various secretagogues using the isolated perfused pancreas of female rats. Insulin release from the perfused pancreas in response to 16.7mM glucose in 8-month-old rats (older rats) was much less than that in 2-month-old rats (young rats). The first phase of insulin release after glucose stimulation was attenuated in older rats. The addition of 0.1mM 3-isobutyl-1-methylxanthine (IBMX) protentiated glucose-inducedinsulin secretion in both groups of rats. However, the second phase of insulin secretion in older rats was lower than that in younger rats. The phorbol ester 12-0-tetradecanoyl phorbol ester (TPA, 200nM) enhanced both the first and the second phases of insulin release induced by glucose in both groups of rats. The amount of first phase insulin release induced by TPA with glucose in young rats was greaterthan that in older rats, whereas the second phase of insulin release was similar in both groups of rats. On the other hand tolbutamide (200uM) similarly stimulated the first phase of insulin release in both age groups of rat. In addition, the amount of cumulative insulin secretion induced by tolbutamide during the second phasc was slightly but significantly greater in older rats than in young controls. Insulin content in the pancreas was significantly greater in older rats than in young rats and increased after the stimulation with TPA and tolbutamide. It is suggested, therefore, that the insulin release machinery in the β-cells is partly hampered by aging, whereas insulin biosynthesis is not impaired in aged rats.

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We studied the anterior pituitary function in 36 patients (25 females and 11 males, mean age: 35 years) with untreated Cushing's syndrome by simultaneous triple stimulus with insulin, TRH and LHRH. Thirty-one patients (86%) had Cushing's disease and five (14%) had an adrenal adenoma. We observed a lack of response of GH to hypoglycemia in 88%, TSH to TRH in 91%, LH to LHRH in 30%, FSH to LHRH in 12% and PRL to TRH in 6% of the patients. Low-to-normal total thyroxine (T4) values were obtained in 37%, with low triiodothyronine levels in 87%. The free-T4 index was normal in all patients. Total testosterone was low in only one adult man, while estradiol and progesterone were low in 45% and 15% of premenopausal women, respectively. We observed no differences in either axis among patients with Cushing's syndrome of different etiologies. Nor was there any statistical difference between the frequency of alteration of each axis and the levels of urinary free cortisol or the duration of the disease. We conclude that hypercortisolism is responsible for the abnormalities in anterior pituitary function in Cushing's syndrome.

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A 77-year-old man was admitted to the hospital with a thyroid nodule. The levels of serum tumor-associated carbohydrate antigens (CA 50, CA 19-9) and thyroglobulin (HTG) were markedly increased. We performed total thyroidectomy and right neck lymph node dissection. After treatment, the serum CA 50, CA 19-9 and HTG levels were markedly decreased. Histological examination of the thyroid tumor showed papillary adenocarcinoma and the dissected neck lymph nodes contained metastatic adenocarcinoma. The expression of CA 50 and CA 19-9 (defined by the monoclonal antibodies) was studied by immunoperoxidase staining from the normal and carcinomatous thyroid tissues and the dissected neck lymph node. CA 50 was expressed more strongly by the carcinoma cells than CA 19-9. The positive rates for serum CA 50 and CA 19-9 levels in other patients with papillary adenocarcinoma were not significantly higher compared with patients with benign nodules and normal subjects. But a significant positive correlation was found between the diameter of the carcinoma and the serum levels of CA 50 and CA 19-9. These results suggest that the serum levels of CA 50 and CA 19-9 might not become useful markers for diagnosing carcinoma of the thyroid, but might be useful markers for monitoring the growth or recurrence of papillary adenocarcinoma of the thyroid in patients with high serum levels of CA 50 and CA 19-9.

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Partial growth hormone deficiency was found in a 13-year-old Japanese boy who was born by breech delivery. Magnetic resonance imaging showed transection of the pituitary stalk and the presence of an ectopic posterior lobe located at the proximal stump of the transected stalk.

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