A radioimmunoassay using the antiserum of prednisolone-3-oxime-BSA according to Erlanger's method is described in this paper. Antiserum produced by immunizing rabbits with prednisolone-3-oxime-BSA conjugates shows a high affinity for prednisolone (K=4.2 × 10⁹ l/mole) and a higher selectivity (the cross-reactivity to cortisol, 17α-hydroxyprogesterone, 21-deoxycortisol and 11-deoxycortisol are 17%, 1.2%, 1% and 1%, respectively) than the antiserum against prednisolone-21-HS-BSA reported by others during the past few years. The best standard curve is obtained with the antiserum diluted 1: 10000-30000 and is established with a useful range between an 0 and 500pg/tube. A small aliquote of the extracted sample is enough for this measurement. The procedure of plasma purification requires a three-step extraction: the first with dichlormethane, the second according to Rudd's method, and the third with a Sephadex LH-20 microcolumn chromatography. The column condition is 25-30°C temperature, and the solvent system is dichlormethane: ethanol:methanol = 97:1:2. The intra- and inter-column coefficients of variation are less than 5.9% when the fraction is obtained from 4.5ml to 6.5ml, with recovery of more than 80%. This method is sensitive to 6ng/ml of 0.1ml plasma. In this method, intra-assay variability of plasma prednisolone determinations is less than 4.1%, inter-assay variability is less than 9.6%. The contamination of endogenous steroids is 1.4 ± 1.62ng/ml (n=37) in normal controls, and 0.65 ± 0.88ng/ml (n=14) in several adrenal disorders. Following oral dosage (1mg/kg/dosage) with prednisolone, the plasma peak values (593 ± 63ng/ml) occurred lhr after the administration, then plasma levels came down to 163 ± 49ng/ml at 6hrs, with the plasma half life of 2.76 ± 0.86hrs. The correlation between the plasma peak values and the dosage in the range from 0.5mg/kg/dosage to 4mg/kg/dosage is r=0.996, p<0.001 in the same person. According to the above results, this prednisolone radioimmunoassay is accurate, precise, sensitive, specific and suitable for routine clinical monitoring of prednisolone levels. Plasma prednisolone measurement may also be of especial value in children who have been given a higher dosage than adults.

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Pharmacokinetics of prednisolone (PSL) were studied in overnight-fasted children following iv. and oral dosage of PSL or PSL-21-hemisuccinate sodium solution (PSL-HS-Na). The evaluation were performed in the aspect of the physiological or pathological influences on plasma PSL metabolism and the effects of plasma PSL metabolism on the hypothalamo-pituitary-adrenal (HPA) axis. The result were as follows.
1. A good correlationship (r=0.92, p<0.001) between plasma peak value (Cp) and single oral dosage was obtained. 2. The dietary (milk 200ml) effect lowered Cp (p<0.001), prolonged late half-life (Tb), and remained unchanged in the area under the concentrationtime curve (AUC) divided by dosage (mg/kg or mg/m2) (unit dosage). 3. Pharmacokinetic studies on five adult volunteers after single 2.5% PSL ethanol iv. administration resulted 83±11 (l/m2/day) in the metabolic clearance rate (MCR), 4.8±0.6 (l/m2) in the central distribution volume (Vc), 5.5 ± 4.0 (l/m2) in tissue compartment of Vc (Vt), and 1.77 ± 0.2 (hr) in Tb. 4. Information obtained from the plasma monitoring after a single oral (lmg/kg) dosage in 20 children and adult controls, who were normal as far as PSL-metabolism, were analyzed for the physiological effect on PSL metabolism. The lower Cp(p<0.01-001), the lower AUC/unit dosage (p<0.01-0.001), higher MCR (p<0.1-0.05) under the 10y/o group, and the longer Tb (p<0.1) in the 1-5y/o group than in the adult group were found statistically in children, which may represent clinically poor absorption, high metabolism and a large distribution volume in children, This may be the reason why the dosage requirement in children of PSL is sometimes greater when the drug is given orally. In contrast, no differences were found between the adult group and in the group after 10y/o, which suggests that PSL metabolism and absorption is developing and maturing. 5. The studies of plasma PSL metabolism under several pathological conditions indicated low Cp, low AUC/unit dosage, slightly high MCR in nephrotic syndrome, prolonged Tb in liver disease, low Cp with low AUC/unit dosage in protein-losing enteropathy, prolonged Tb, increased Vc, low MCR in bilateral nephrectomized rats and cardiac-failed dogs with bilateral femoral A-V bypass operation. 6. Simultaneous monitoring on blood ACTH, cortisol (F), 17-OH-progesterone (17 OHP) and PSL were performed in 14-adrenal disorders in order to investigate PSL-maintenance dosage-effect on the HPA axis. Three patterns of response to PSL in HPA suppression were divided in various ways, such as good suppression with normal PSL levels (n=10, 53 ± 7ng/ml), poor suppression with poor PSL levels (n= 2;33, 35ng/ml), poor suppression with normal PSL levels (n=2;5 0, 57ng/ml). One of the second pattern-cases improved after a double dosage to good suppression with good PSL levels (57ng/ml). These findings suggest that plasma PSL levels around 40-50ng/ml may be the borderline level in HPA suppression. Upon analyzing these poor responders, half of them showed poor intestinal absorption, while another half of them suggested some ab-normality in HPA regulation mechanism. One case of 21-OH-lase deficiency developed another pattern of insufficient suppression of HPA aixs after a single oral F-maintenance dosage (10mg/m2/dose), (poor suppression of 17 OHP with good suppressed ACTH and normal F level (382ng/ml)), which showed it was important for the evaluation of HPA suppression to know the adrenal condition (adrenal hyperplasia, etc.). To monitor plasma PSL levels, if the individual PSL-adsorption and metabolism may be influenced by physio-logical and pathological factors, it is important and useful to know the achievement of PSL treatment, especially in children.

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Selective adenomectomy of patients with Cushing's disease results in marked clinical improvement and recovery of normal pituitary-adrenal function, including diurnal rhythm. These findings tend to exclude a hypothalamic origin of Cushing's disease; nevertheless its etiology is controversial.
The present in in vitro study was designed to investigate the autonomy of ACTH secretion from the adenoma under the absence of a hypothalamic regulatory mechanism. Fragments of the pituitary adenomas from seven patients with Cushing's disease were placed in primary culture immediately after surgery. Hormonal release was studied from two weeks to three months depending upon the survival culture. Adenoma tissue was proved to release continuously a high concentration of ACTH into the medium for a month. ACTH was still detectable in one case even after three months. Contrarily, ACTH secretion from residual anterior pituitary tissue in Cushing's disease and normal pituitary decreased rapidly and became undetectable within a few weeks in vitro.
In addition to the explant culture study, the effect of lysine-vasopressin (LVP) on ACTH secretion from perfused adenoma tissue was examined, since clinical data in the ACTH response to LVP before and after surgery argues that the conspicuous preoperative response is caused by adenoma. ACTH secretion was induced with little or no latency after LVP infusion in seven out of eight cases. However, a dose-response relationship was not obtained between the LVP concentrations and the magnitude of ACTH responses. The in-fusion of dexamethasone, on the other hand, inhibited ACTH secretion in only one of four cases.
These data suggest that the autonomy of ACTH secretion may exist in the adenoma of Cushing's disease and that LVP acts directly on adenoma tissue such as proposed by clinical data.
From our clinical results and experimental studies in vitro, it can be concluded that the primary lesion in Cushing's disease seems to be in the pituitary gland rather than in the hypothalamus.

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Relationships between plasma cAMP concentration, cAMP content in various tissues and urinary cAMP excretion were investigated to elucidate cAMP metabolism in streptzotocin-induced diabetic rats. The rats were made diabetic by a single injection of 60mg/kg of streptozotocin into the cervical vein. The plasma cAMP in various blood vessels and cAMP content in various tissues were measured 10 days after the injection of streptozotocin. Urinary cAMP excretion levels were determined 5 days before to 10 days after the streptozotocin injection.
The increase in plasma glucose and IRG and the decrease in plasma IRI were certified in diabetic rats. The plasma cAMP concentration in the cervical vein and abdominal aorta in diabetic rats did not change significantly although their plasma cAMP concentration in the hepatic vein was significantly increased compared with the control rats. Hepatic cAMP content in diabetic rats was higher than that in the control rats, but the cAMP content in the renal cortex in the diabetic rats was significantly lower than that in the control rats. In the diabetic rats, the cAMP content in the brain, myocardium, small intestine and skeletal muscle was not different from that in the control rats.
The urinary cAMP excretion level in the diabetic rats was significantly higher than that in the control rats from the second day to the 10th day after the injection of streptozotocin. The creatinine clearance as well as the cAMP clearance in the diabetic rats was higher than that in the control rats.
In conclusion, in streptozotocin-induced diabetic rats, the plasma cAMP concentration did not increase after the injection of streptozotocin, despite an increase in cAMP content in the liver and plasma cAMP level in the hepatic vein. The increase in urinary cAMP excretion level in the diabetic rats as a result of increased glomerular filtration may be one reason for the failure of plasma cAMP to increase.

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[in Japanese]

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Distribution of immunoreactive LHRH-like substance was studied in the brains of 5 species of mammals, 1 species of birds, 7 species of reptiles, 7 species of amphibians, 6 species of teleosts, 1 species of elasmobranchs, and 2 species of cyclostomes. For this pur-pose, two different anti-synthetic LHRH sera (AsI : anti-Tyr₅-BSA-LHRH serum; AsII : anti-Gly₁₀-BSA-LHRH serum) were used.
Among mammals and birds, LHRH-positive perikarya were observed by both AsI and AsII in the septo-preoptic region of the cat, and by AsI in several regions of the fore-brain including the septo-preoptic region of the guinea pig. However, they were not detected in the brains of the rat, mouse, hamster and Japanese quail in either case of AsI or AsII. In any case mentioned above, LHRH-positive material was found in the nerve terminals of the median eminence.
In reptiles and amphibians, similar results were obtained by both AsI and AsII. LHRH-positive perikarya were generally distributed in the septo-preoptic region, and their nerve terminals were consistently detected in the median eminence. Only in Xenopus, however, LHRH-positive perikarya were distributed in the peri-chiasmatic region, the infundibular region, the anterior part of the optic tectum and the olfactory bulb, in addition to the septo-preoptic region. In most species of reptiles and amphibians, intra- and extra-hypothalamic LHRH-pathways, which proceed toward (1) the median eminence, (2) the olfactory bulb, (3) the optic lobe, and (4) the lower brain stem were demonstrated.
LHRH-positive material was detected by both AsI and AsII in the neurohypophyses of the eel, goldfish and puffer, but only by AsI in the lamprey. Among the former three species, only in the eel, LHRH-positive perikarya were observed : they were distributed in the ventro-medial part of the telencephalon and the olfactory bulb, and they projected their fibers mainly to the anterior portion of the neurohypophysis. In the lamprey, those perikarya were distributed only in the preoptic nucleus. LHRH-positive material was detected by both AsI and AsII in the brain of the dogfish. However, distribution of those fibers were quite different from other species mentioned above, and no LHRH-positive terminals were detected in the median eminence. In the brain of the rainbow trout, loach, medaka and hagfish, LHRH substance was not detected by either AsI or AsII.

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