Crude ACTH preparation (Cortrophine) was selected as the starting material for the present studies. Crude ACTH preparation was subjected to gel filtration at 4°C on a Bio-Gel P-10 column of 1.5 × 85 cm. The column was eluted with 0.1N acetic acid, and 3 ml eluates were collected. Immunoreactive ACTH contents in each fraction were determined by radio-immunoassay. Bioassay was performed using isolated rat adrenal cells. On gel filtration, several immunoreactive and bioactive ACTH components were separated. These peaks were arbitrarily designated Fraction A and C. Fraction A emerged earliest, hence was assumed as having a larger molecular size. Fraction C was eluted at a position closely corresponding to that of authentic porcine ACTH. When Fraction A and C were refractionated on the same column, both of them emerged as homogenous peaks with no shift in their respective elution patterns. The immunoreactivity of Fraction A and C after refractionation were tested with an antiporcine ACTH antibody. Fraction C was found to have close immunologic similarity with authentic ACTH, where Fraction A seemed to have somewhat different immunor-reactivity. There was reasonable agreement between the biologic and immunologic potencies of Fraction C, when assayed using authentic or synthetic ACTH as a standard.
In contrast, the biological activity of Fraction A was less than 1% of the activity as measured by radioimmunoassay. The biological activity of Fraction A, however, was not negligible, since it was clearly demonstrable when a considerable amount of it was subjected to the assay using isolated rat adrenal cells.

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副腎遊離細胞によるACTHのbioassayを用いた著者らの検討改変による, in vitroのACTH放出活性測定法を考案して, 活性アミンの下垂体レベル直接作用にもとづくACTH放出活性を検討し, 次の成績を得た.
1) NA (4×10^-4M), 5HT (3.3×10^-5M), DA (2×10^-4M) で各々対照の234%, 249%, 160%とACTH放出活性を認めた.
2) これらの作用はレセルピン前処置ラット下垂体を用いると, NAでは4×10^-5Mでも約200%とACTH放出が高まるのに反し, 5HT, DAでは上記濃度でも活性が認められなくなった.
3) NAの作用はβ blocker, propranololにて, 5HTの作用はmethysergideの併用によって抑制され, それぞれβ receptor及びserotonergic receptorを介する特異的なものと考えられる.
4) NA, 5HTのACTH放出作用に対する, DXの下垂体レベルでのnegative feedback効果はin vitroで証明されなかった.
5) phentolamine, propranolol, methysergide及びDXは, それぞれ単独では, ACTH放出に影響を与えなかった.

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In the first report, we demonstrated that the biogenic amines noradrenaline (NA), dopamine (DA) and serotonin (5HT), can act directly on the rat anterior pituitary to increase ACTH release in vitro, using a revised in vitro method for the assay of corticotropin releasing factor (CRF) activity developed in our laboratory. Cyclic 3', 5' adenosine monophosphate (cyclic AMP) has been implicated as a mediator of many hormonal actions, including the effect of hypothalamic releasing hormones on the release of pituitary hormones. Therefore, ACTH release induced by biogenic amines in vitro may be mediated through cyclic AMP. Based on these considerations, we studied the effect of biogenic amines on the cyclic AMP content in the rat anterior pituitary and considered the correlation of ACTH release and cyclic AMP increase stimulated in vitro by biogenic amines. Furthermore, we also considered the correspondence between the changes in the two parameters induced by some amine inhibitors and dexamethasone (DX).
After two paired rat hemipituitaries were preincubated in 2 ml of Krebs-Ringer bicarbonate buffer (KRB) containing 0.2% glucose and 10^-2M theophylline at 37°C for 30min. under a moist atmosphere of 95% 02 and 5% CO₂, the medium was replaced with 2 ml of fresh medium containing the test substances and ascorbic acid (1 mg/ml) to prevent oxidative degradation of amines. Incubation was then carried out for an additional 15 min. Paired controls were employed throughout. The incubation was terminated by freezing the incubated pituitaries on alminium foil immersed in dry ice and acetone. Frozen tissues were homogenized in ice cold 6% trichloroacetic acid and denatured protein was removed by centrifugation. Precipitates were dissolved in 1 N NaOH and assayed for protein by Lowry's method⁸). Supernatants were washed with cold water-saturated ether and cyclic AMP was determined by the competitive protein binding assay of Gilman⁶) using the binding protein obtained from rat liver according to the method of Kumon et al.⁵) or by the radioimmunoassay of Steiner et al.⁷) Both methods gave comparable values using the same tissue. The results were expressed as nanomoles/gram of tissue protein or percentage of paired control. The system and method for measuring ACTH releasing activity were the same as described previously¹). The paired comparison Student's t test was used for statistical analysis.
The following results were obtained :
1) NA increased cyclic AMP content in the pituitary at a concentration of 4 × 10^-4M which was the same concentration at which it stimulated ACTH release from the pituitary in vitro. However, it was ineffective at 4 × 10^-5M, which was also the case in stimulating ACTH release. Furthermore, when reserpine pretreated rat pituitaries were used, (17 hrs before decapitation, 1 mg/rat, i.p.) a concentration of 4 × 10^-5M of NA, which was capable of stimulating ACTH release, also increased pituitary cyclic AMP content. These effects of NA on both pituitary ACTH release and cyclic AMP content were inhibited by the β blocker propranolol, but not by the a blocker phentolamine. In spite of addition from the preincubation period, DX had no effect on either increasing ACTH release or cyclic AMP in response to NA.
2) 5HT increased both pituitary ACTH release and cyclic AMP at a concentration of 3.3 × 10^-4M. Even at a lower concentration (3.3 × 10^-5M), 5HT stimulated the ACTH release significantly and was also found to tend to increase the cyclic AMP content to 145% of that of the control although this was not significant. These effects of 5HT on both pituitary ACTH release and cyclic AMP content were inhibited by the same concentration of the serotonergic blocker methysergide,

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We have developed a clinically useful enzyme-labelled immunoassay (EIA) for plasma cortisol. Alkaline-phosphatase (AL-P) from E. coli was used as a tracer. Water soluble carbodiimide was used as a conjugating reagent between enzyme and cortisol-21-hemisuccinate. Cortisol-AL-P conjugate was separated from unreacted cortisol by means of a Sephadex G-100 column. Anti-cortisol-21-hemisuccinate BSA rabbit serum was used in this system, however anti-cortisol-3-oxime BSA rabbit serum did not work in this system. 10 μl of plasma was used for assay after denaturation of endogenous cortisol binding globulin and AL-P by heating in a glutamate buffer. B-F separation was done by means of the double antibody method. AL-P activity of antigen-antibody complex was measured by means of the Kind-King method.
Sensitivity, specificity and measurable range of the present EIA method were quite similar to those of the RIA method using ¹²⁵I-cortisol or ³H-cortisol. This method might be useful and generally available for routine analysis of plasma cortisol because the use of radioactive materials is avoided.

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甲状腺組織の形態を保持したまま, ヨウ素や電解質およびタンパク質の分布・存在状態を知る目的で, X線マイクロアナリシス法を試みた.基礎的条件の検討では, 凍結切片またはそれをアルコール固定した方法が目的に適することを見出し, 次いで生理的状態の異るブタ甲状腺の比較をおこなった.抗甲状腺剤投与でNa, K濃度が著しく減少すること, KI投与とKCl投与とでは, 甲状腺におけるKの蓄積に差があることなどを見出した.

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The interaction of insulin and insulin receptor was studied with epididymal fat cell membrane of Zucker fatty rats and compared with their thin littermates or wild type normal rats. Epididymal fat pads were removed from Zucker fatty rats (10-11 months old, body weight 600-700 g) and from thin littermates or normal rats (9-12 months old, body weight about 300 g). Purified plasma membranes were obtained by the Rodbell's method.
Prepared cell membranes (130 μg protein) were incubated with ¹²⁵I-insulin (about 40 μU, specific activity 97 μCi/μg) and native insulin (0-20 μg) or native proinsulin (0-10 μg) in 0.1% bovine serum albumin Krebs-Ringer bicarbonate buffer (pH=7.4). Final incubation volume was 0.6 ml per tube. Incubation was performed for 20 minutes at 24°C. Labelled and native insulin bound to fat cell membrane were separated from free insulin by filtration through a millipore filter. Filters were washed with 5 ml of chilled Krebs-Ringer-bicarbonate buffer and the specifically bound radioactivity was counted.
The following results were obtained :
1) The insulin binding capacity per mg protein of epididymal fat cell membranes from Zucker fatty rats was about 40% of that from control rats.
2) There were two or more receptor sites on fat cell membranes from both Zucker fatty rats and control rats. The equilibrium constant (Km) of high affinity site was about 2.9-4.4 × 10^-9 mol/1.
3) In Zucker fatty rats, inhibiting effect of unlabelled proinsulin on the binding of ¹²⁵ I-insulin to fat cell membrane was slightly more potent than in the control rats.
4) The specific insulin binding capacity on cell membrane was readily altered by medium glucose concentration in all strains of rats.

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The correlation between insulin-binding to receptor and biological activity of bound insulin was simultaneously studied with the same epididymal fat cells of Zucker fatty rats and their thin littermates or wild type normal rats. Fat cells were prepared from the Zucker fatty rats (10-11 months old, body weight 600-700 g) and from their thin littermates or normal rats (9-12 months old, body weight about 300 g). The amount of bound insulin and the rate of glucose oxidation were determined in the same isolated fat cells after incubation with various concentrations of ¹²⁵I-insulin with the constant specific activity for estimation of insulin dose response, or with U-¹⁴C-glucose of increasing carrier glucose concentration, in order to quantitate the glucose effect. Cells were equilibrated with 95% O₂ : 5% CO₂ and incubated for one hour at 37°C in Krebs-Ringer-bicarbonate buffer without bovine serum albumin (pH=7.4). The insulin bound to fat cells were separated from free insulin by isolation of fat cells. The biological activity was estimated by measuring the rate of conversion of U-¹⁴C-glucose into ¹⁴CO₂ or ¹⁴C-Lipid per cell per hour.
The following results were obtained.
1) The glucose conversion to CO₂ increased proportionally to medium glucose concentration on fat cells of both fatty and control rats. The dose response of glucose oxidation rate was greater in the Zucker fatty rat cells than in the control rat cells in use increasing concentration of medium glucose. On the other hand, when medium insulin concentration was increased, the proportional increment of glucose conversion to CO₂ was not so extentive in fat cells of Zucker fatty rats as in the control fat cells.
2) On Zucker fatty rats and control rats, the conversion rate of glucose into CO₂, as an index of the biological activity of insulin, was not proportional to the amount of bound insulin.
3) Insulin binding capacity per cell in Zucker fatty rats was not lower than in control rats under the biological range of glucose and insulin concentration.
In conclusion, mechanism of insulin resistance in Zucker fatty rats should be partially attributed to the abnormality of intracellular steps of glucose metabolism.

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The first successful analysis of TRH in serum and urine was performed by mass fragmentography using a GC-MS combined system. In developing our method, the first problem we had to solve was that of forming a volatile, stable derivative of TRH. TRH was methylated by addition of ethereal-alcoholic solution of diazomethane. This derivative was most useful for GLC work.
The equipment used was Shimadzu LKB 9000 GC-MS (MID-PM). The methylated derivative of TRH was analyzed using the GC-MS system equipped with a 3 ft × 3 mm column packed with 1.5% OV-1 and the temperature was programed at 260°C.
The mass spectrum showed molecular ion at m/e 390 which corresponds to the dimethyl derivative of TRH. The base peak at m/e 149 was applied to establish the precise quantitative evaluation of TRH and related compounds by mass fragmentography.
Inactivation of TRH by serum and urine was well documented. Urines were collected and kept at pH 3 over a 24-hr period. Specimens were stored at -20°C until extraction and assay. Blood samples were collected into polystyrene tubes containing 5mg BAL (2, 3- dimercaptopropanol) and kept in chilled ice. After centrifugation at +4°C, serum was separated and stored at -20°C.
The following results were obtained :
1) The sensitivity was of the order of ng or pg which enables quantitation with 2ml of human serum and urine sample.
2) The procedure is specific, sensitive, reproducible and rapid.
3) The use of GC-MS system to analyze hypophysiotropic releasing hormones enables us to elucidate the relative importance of these hormones and their metabolic changes in various physiological and pathological states of human beings.

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When chlormadinone acetate, an anti-androgenic agent, is administered to rats and humans, a significant fall in plasma testosterone levels is demonstrated during the treatment. In connection with the decrease of plasma testosterone the biosynthesis of testosterone in the testes and metabolism of testosterone in the liver was investigated in rats treated with chlormadinone acetate. When 100 mg/kg of chlormadinone acetate were administered to rats for 3 and 5 weeks, conversion ratios of testosterone to 5α-dihydrotestosterone and 5a-androstandediol in the liver were 2-3.5 fold greater than the control. While, under similar conditions hydroxylation of testosterone (2α-, 6β-, 16α-hydroxylation) decreased to the extent of 50% of the control. Further, the 5α-reductase activity rose to about 3 fold by the treatment of estradiol 3-benzoate (0.4 mg/kg) for 5 weeks. On the other hand, no effects of chlormadinone acetate on hepatic 5α-reduction and hydroxylation of testosterone were observed by the removal of pituitary gland. These findings indicated an essential role of the pituitary gland in androgenic control of hepatic enzyme.
While, the homogenate of testes from rats treated with chlormadinone acetate for different periods was incubated with progesterone-4-¹⁴C and cholesterol-4-¹⁴C, and assayed for conversion rate of the substrates to testosterone. Biosynthesis of testosterone from cholesterol-4-¹⁴C in rat testes treated with chlormadinone acetate for 4 weeks showed a marked inhibition depending on the dose of chlormadinone acetate, varying from 40% to 50% with 20 mg/kg to 100 mg/kg of chlormadinone acetate. While the conversion rate of progesterone-4-¹⁴ C to testosterone was inhibited to the extent of 16% by chlormadinone acetate treatment (100 mg/kg) for 4 weeks, no effects with 20 mg/kg of chlormadinone acetate and 3β-hydroxy chlormadinone acetate, one of the main metabolites in human and rat.
It is possible that reduction of plasma testosterone levels observed during chlormadinone acetate treatment is ascribed to suppression of biosynthesis of testosterone in the testes and to stimulation of the metabolism in the liver.

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