Hellerström (1964) described a method of the microdissection of islets in the mouse.
Insulin can be destroyed by lytic substances released from the acinar tissues, but it may be possible that secreted insulin from microdissected pancreatic islets is less exposed to the lytic effects of any acinar tissue.
The purpose of the present communication is to demonstrate a new approach to the mechanism of insulin synthesis and release from mammalian islets.
It was reported that islets isolated by this procedure have been used for in vitro studies on enzymatic activity and oxygen consumption of individual islets.
We have already previously reported that insulin secretion of one microdissected islet by stimulation of hexoses and hormones was measured by radio-immunoassay.
In this investigation we have studied the effects of exogenous insulin on endogenous insulin release from microdissected islets of mice.

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In an attempt to find out whether there are differences in character among the HCG of early pregnancy, late pregnancy, hydatidiform mole and chorioepithelioma, and also to clarify the relationship between immunological and biological activity of HCG, this investigation was performed as follows. Crude urinary extracts of normal pregnancy, hydatidiform mole and chorioepithelioma were prepared by the Kaolin-ethanol method and analyzed by Sephadex gel filtration and DEAE-C chromatography. The biological activity of HCG in each fraction was estimated by rat ovarian weight and the immunological activity by HAIR. The titer of antiserum used in HAIR was 1 : 16,000 according to the method of Wide. One ml antiserum absorbed with 15 mg of urinary protein did not make a precipitate line with both urinary protein and serum protein in gel diffusion plate. The antigen used in HAIR was sensitized to the catalase fixed red cell of ox by the B.D.B. method. On analysis by Sephadex gel filtration, both biological and immunological activities were high in the same fractions in the HCG of normal pregnancy, while the fractions of high immunological activity was only a part of fractions of high biological activity in the HCG of chorionic tumors. On analysis by DEAE-C chromatography, in all four HCG, the high immunological activity was only in the fast eluted fractions of high biological activity. Comparing the HCG of hydatidiform mole with the HCG of late pregnancy, the immunological activity was lower than the biological activity. In the inactivation experiments of high purified HCG (12,000 I.U./mg), the immunological activity of HCG was not changed by incubation at 37°C or 50°C for 6 hours, but decreased to 25% by boiling 30 minutes. By incubation with urea, varitase, LAP or CPase, there was no decrease in immunological activity but 50% decrease was noted by incubation with RDE or α-chymotrypsin. The urinary HCG concentration in normal pregnancy, estimated by HAIR, was 88.9 I.U./ml in the 3rd month, 5.6 I.U./ml in the 7th month and 10.4-12.5 I.U./ml in the 8-10th month respectively. In a case of early pregnancy, the urinary HCG concentration amounted to 256 I.U./ml in the 8th week, 256-64 I.U./ml during the course of 8th to 15th week, then 64-3 I.U./ml during the course of 16th to 22th week, respectively. There were considerably large daily changes. In a case of hydatidiform mole, the HCG concentration of molar cyst fluid was 5-6 times higher than that of urine at molar delivery.

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Estradio1-17β metabolism in the various breast tissues from the patients with mastopathy (16), fibroadenoma (7), breast cancer (38) and the others (23) were studied.
The tissue slices from the patients were aerobically incubated in vitro with estradiol-17β-4-C¹⁴ (31.8 mc/mM), and the metabolism of estradio1-17β was compared with that of estrone-4-C¹⁴ (29.5 mc/mM) and testosterone-4-C¹⁴ (21.7 mc/mM). Two metabolites were observed from estradio1-17β. Estrone was the major metabolite, which was identified by chromatography on paper and silicagel, derivative formation, and recrystallization to constant specific activity. The other minor metabolite might be estriol as judged from the results of the chromatographic experiment.
It was shown that there was no qualitative difference in the metabolism of these tissues studied. These tissues, however, showed quantitatively different results in the formation of estrone. The mean values of production rate of estrone was higher in mastopathy, lactation's breast and metastatic breast cancer tissues and lower in the primary breast cancer tissues than normal breast tissues. It was found that this difference was not entirely due to the cellularity of tissues in the various patients. In general, the production rate of estrone was low in both malignant and non-malignant tissues from old patients.
An analysis of the relationship between histological findings and estradio1-17β metabolisms of breast tumors indicated that the tumors classified histologically as comedocarcinoma, adenocarcinoma papillotubulare, and medullary carcinoma had high production of estrone and the tumor classified as scirrhous carcinoma had low production. It was also found that the production of estrone was lower in the lower degree of tissue differentiation.
In the metabolisms of estradio1-17β and testosterone it was found that the more estrone was formed from estradio1-17β, the more androst-4-ene-3.17-dione was formed from testosterone by the same tissue. Moreover, the metabolism in cancerous tissue was compared with that in non-cancerous tissue from the same patient. These results indicated that the cancer appeared less active in the formation of metabolites.
Finally it was found that testosterone inhibited the formation of estrone from estradiol-17β at the concentration of 10^-3 M, 10^-5 M, and that the reverse reaction (estrone→ estradiol) was much lower than the forward reaction (estradiol→estrone) in the same tissues.

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It seems that the blood sugar level may be affected by many factors which give various features to the clinical manifestations and therapeutic responses of diabetic patients. The authors tried to study the relationship between the insulin sensitivity and clinical characteristics in 405 hospitalized diabetic patients in our hospital in these three years.
Insulin sensitivity test were performed as follow :
Ten units of regular insulin was injected subcutaneously early in the morning, and blood sugar levels were determined by Hagedorn's method before and 1, 2, 3, 4, 5, 6 hours after insulin injection. Insulin effect and insulin rebound effect were calculated by following formulas.
Insulin effect=a-b/a × 100 (%)
Rebound effect=c-b/a × 100 (%)
a : Blood sugar (mg/dl) before insulin injection.
b : The lowest blood sugar level after injection.
c : Terminal level of blood sugar.
Mean values of insulin effect and rebound effect in this study were 51.5% and 23.2% respectively.
All patients were divided into next five groups (A, A', B, C and D) according to their results of insulin sensitivity test (Fig. 1).
1. A group : normal insulin effect and normal rebound effect. 216 cases (53.3%)
2. A' group : increased insulin effect with non-rebound effect. 57 cases (14.1%)
3. B group : decreased insulin effect with normal rebound effect. 50 cases (12.3%)
4. C group : normal or increased insulin effect with high rebound effect. 72 cases (17.8%)
5. D group : decreased insulin effect with high rebound effect. 10 cases (2.5%)
We called B group as “insulin resistant group”, considering the presence of a possible factor to inhibit insulin action, and C group as “insulin rebound group”.
The results of our investigation of the clinical characteristics of these groups were as follows.
(1) Many severe diabetic cases were found in both of A and A' groups. (Table 2)
(2) Good responses to the treatments were observed in only the patients in A group, and in another patients, the treatments seem to be less effective. (Table 3)
(3) In A group both of insulin treatment and SU treatment were effective, but in A' group only insulin was effective, and in rest other groups, SU was effective. (Table 4, 5)
(4) Many insulin dependent diabetics were found in A' group and they showed high ketotic tendencies.
(5) From the view of accompanying diseases, liver dysfunction was popular in B and C group and retinopathy in A and C groups, but hypercholesteremia was found equally among all groups.
(6) Changes in serum protein content and albumin-globulin ratio (A : G ratio) after insulin injection were observed and following three types of restoration mode were found (Fig. 2).
I Complete restoration type :
A complete restoration was shown both in serum protein content and in A : G ratio. Many cases belonging to this type were found to be in C group.
II Incomplete restoration type :
Either serum protein content or A : G ratio got a restoration in this type. Many cases belonging to this type were found to be A group.
III Defective type :
Neither serum protein content nor A : G ratio got a restoration in this type. To this type, B group cases and partial A' group cases belonged. Responses to the treatments were seemed to be very poor in these cases.
In short, there would be some peculiar characters in the groups showing abnormal insulin sensitivities (A', B, C and D). In C group, antagonistic hormones to insulin may have a strong activity, but in A' group the activities of both of insulin and antagonistics to insulin are extremely decreased.
In B group, a decreased activity of insulin or a increased activity of insulin antagonists is presumed, and D group may possess both characters of B and C groups.

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It has been suggested that estrogen might counteract the haemorrhage of various origins, the development of atherosclerosis and induced local edemas. Numerous evidences have been demonstrated that estrogen has a suppressing influence on vascular permeability. However, many questions remain regarding its mechanism. In order to analyse the mechanism, the author has performed this experiment to study the effect of estrogen on the vascular permeability-increasing effect of bradykinin and histamine.

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Fractionation and identification of cortisol in the plasma, using the steroid binding properties of competitive protein binding employing Gel filtration and insoluble adsorbent (Lloyd's reagent) are now widely carried out. Namely, the addition' of an increasing amount of unlabeled cortisol to an equilibrium dialysis system containing standard plasma and a constant amount of cortisol-1, 2-³11 exerts a proportional decrease in the percentage of cortisol-1, 2³-H bound to the plasma protein.
On the other hand, preparations of plasma containing unknown amounts of cortisol also decrease binding. The cortisol content could thus be determined by a standard curve. These methods required 1 ml and 0.1 ml of test plasma and gave a standard deviation of 0~80 midi and 0~40 mμg/ml, respectively. In the present paper the plasma cortisol determinations were carried out on samples from 195 subjects by the method of Gel filtration and Lloyd's reagent. After extracting the cortisol 1 ml or 0.1 ml by ethylalcohol it was measured according to its competition with tritiated cortisol for binding sites on the corticosteroid binding globulin of female plasma. The concentration of total cortisol was measured in maternal and cordal plasma, as well as in healthy female subjects and in inpatients by the method of Gel filtration and Lloyd's reagent.
The comparisons of the data by Gel filtration and Lloyd's reagent are shown as follows. Plasma cortisol concentration by the method of Lloyd's reagent was higher than that of Gel filtration except in cordal plasma. Plasma cortisol concentrations in healthy subjects (7.74±3.04 μg/dl) by Gel filtration and (11.21±12.15 μg/dl) by Lloyd's reagent were lower than those in inpatients (11.50±3.68 μg/ di) by Gel filtration and (17.67±2.90μg/dl) by Lloyd's reagent. It was observed that cortisol concentration of cordal plasma (42.64±8.60 μg/dl) by the method of Gel filtration was higher than maternal plasma (34.99±11.81 μg/dl) by the method of Lloyd's reagent. Cortisol concentration in cordal plasma (17.40±16.37 μg/dl) by the method of Lloyd's reagent, however, was lower than that of maternal (39.67±12.00 μg/dl) by the method of Lloyd's reagent.
Cortisol is known to be the corticoid present in the largest amount in the female plasma and its concentration to be increased following the administration of ACTH.
Unbound cortisol levels in the maternal and cordal plasma were higher than those in healthy non-pregnant.
Both unbound and bound cortisol levels in the maternal and cordal plasma increased by chronic stress, but the elevation of cortisol levels in the maternal and cordal plasma was not observed by acute stress. It is generally assumed that the placenta acts as a barrier which allows only maternal cortisol to transfer the fetal circulation.

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In our previous paper, we reported that HCG could be successfully assayed radioimmunologically in only 60 minutes using insoluble rabbit anti-HCG serum polymerized by ethyl chloroformate (Folia Endocr. Japonica, 45 : 556, 1969). We also succeeded in assay-ing HCG using immunoadsorbent which was prepared by polymerizing anti-HCG serum by glutaraldehyde. Three ml. of rabbit anti-HCG serum which was completely absorbed with child urine protein and lyophylized human plasma protein, were diluted with 20 ml. of 0.1 M acetate buffer (pH 4.9). After mixing 500 mg. of cellulose powder MN (Macherey Nagel & Co.) in the diluted anti-HCG, 0.6 ml. of 2.5 M (25%) glutaraldehyde was added to the mixture and stirred gently for 2 hours. The resulting precipitate was ground by Potter-Elvhjem glass homogenizer and washed repeatedly with 400 ml. of saline until the OD₂₈₀ of the supernatant became zero. The washed precipitate was suspended and diluted with borate buffered saline (pH 8.2) to 75 ml., and then kept at 4°C after the addition of merthiolate (1 : 10,000). Before use, the polymer was washed once with borate buffered saline by centrifugation (3,000 r.p.m., 20 min.) and resuspended. As control polymer to examine the nonspecific adsorption of ¹²⁵I-HCG to immunoadsorbent, the normal rabbit serum was polymerized by the same procedure as the anti-HCG serum. The slight antigen excess region was determined for the polymerized anti-HCG, in order to get the most sensitive experimental condition for assay. The binding of ¹²⁵I-HCG to polymerized anti-HCG reached the maximum in 5 hours at 37°C, but 73% of the maximal binding was obtained in 60 minutes at 37°C.
The binding of ¹²⁵I-HCG to polymerized anti-HCG serum was inhibited by the presence of various amounts of cold HCG. From the standard inhibition curve of HCG for binding ¹²⁵I-HCG, 8 mI.U./ml. of HCG could accurately be assayed by 60 minutes reaction.
The procedure of sasay is very simple : Three tenth ml. of specimen (urine, serum or standard HCG), 0.3 ml. of diluted anti-HCG polymer suspension (1 : 200) and 0.002 I.U. of "5I-HCG in 0.3 ml. (ca. 20,000 cpm) were mixed and incubated in the water bath (37°C) with gentle shaking for 60 minutes, and then centrifuged at 3,000 r, p.m. for 20 minutes. The precipitate was washed once with borate buffered saline and the radioactivity of washed precipitate was measured by a scintillation counter.
This is the one step direct reaction between HCG and anti-HCG, therefore it is not necessary to use EDTA for the assay of HCG in the serum as two antibody method. The recoveries of the protein by polymerization were very constant and showed 95.1 % by ethyl chloroformate and 95.4% by glutaraldehyde. The solubilization of polymers during storage is a very important factor for the accuracy of assay. Only 0.7% of polymer by ethyl chloroformate was solubilized during 6 months and 2.3% of the other by glutaraldehyde was solubilized during 50 days. The binding capacities of polymers to ¹²⁵I-HCG by ethyl chloroformate and glutaraldehyde showed more than 50% even after long storage and from the above results it was proved that both polymers were very stable for storage. This rapid radioimmunoassay is not only used to follow up the clinical course of chorionic tumor patients for assaying low titer of HCG, but also used for LH assay using serum of women in our laboratory.

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The urinary free aldosterone determination is one of the tests used for assessing adrenocortical function in clinical medicine.
Several workers have obtained the measurement but these methods are too elaborate and time-consuming. To obtaina rapid and simple method I compared the fluorometric method with the colorimetric determination with B.T.
The fluorescence of aldosterone in strong acid medium is not yet definitely known. Its absorption peak in sulphric acid was obtained at 288 mμ, and after heating the same solution to 100°C for 5 minutes its peak was detected at 340 mμ (Fig. 2, 3).
Because of these results it was thought that the fluorescence of aldosterone in acid medium could be produced.
The dry samples of D-aldosterone at three concentrations, 2, 1 and 0.5 μg per ml, were used.
75% sulpuric acid ethanol (v/v) was used as acid medium.
The measurements were carried out with spectrophoto-fluorometer at an exitation wave length of 470 mp and emission wave length of 520 mμ.
As a result of the study the most suitable temperature and time were obtained by heating aldosterone in sulphuric acid medium at 75°C for 5 minutes (Fig. 4, 5). The fluorescence of the cooled aldosterone solutions proved to be stable for at least 5~25 minutes.
Concentration-fluorescence curve was obtained as a straight line in the range of 0.25~3 μg per ml (Fig. 8). The samples, at least 3 ml of acid medium are required, were examined with the Aminco Bowman fluorophotometer.
The purification of the urinary aldosterone was carried out by the modified method of Oda & Suzuki which uses florisil column, three T.L.C. systems and acetylation (Table 1).
The results of determination on the urines of four normal subjects were higher (average : 25 μg per 24 hrs.) than the normal range (average : 10±3 μg per 24 hrs.) by the colorimetric determination with B.T. (Table 3).
At the same time, a modification of the B.T. method which is based on Oda & Suzuki's method was tried to obtain a rapid and simple technique.
Aldosterone concentration curve by the colorimetric determination with B.T. was obtained as a straight line in the range of 2.5~30 μg (Fig. 11).
500 ml of 24 hrs pooled urines were used, and they were extracted three times with 100 ml chloroform (Table 4).
The excellent recovery rate was average : 50~55%
The excretion of urinary aldosterone was measured by this method, in one patient with hypertension with nonsuppressive plasma renin activity, one case of Conn's syndrome and three healthy adults. Aldosterone values of three normal cases were from 13 to 14 pg per 24 hrs., hypertensive case was 33 μg per 24 hrs. and Conn's syndrome was 17 μg per 24 hrs. (Table 5).
The results of the fluorimetric method have been compared with those of the B.T. method on several urinary samples. In spite of the purification of the urinary aldosterone at several stages, the relatively high and unstable data were obtained in four normal human urines by the fluorimetric method (Table 3). It was thought that the fluorescence of several unknown materials interacted for the determination of urinary aldosterone. The result of the B.T. method was better than that of the fluorimetric method. For instance, the B.T. method was relatively quickly performed and the measurement data of urinary excretion of aldosterone were exact in a variety of clinical circumstances.

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In order to demonstrate the major role of thyroid function in the cold environment, plasma Thyroid-Stimulating-Hormone (TSH) levels in cold environment were investigated and the following observations were obtained.
Animals; Male rats (Weight ca 100 gm, Swiss-Webster strain) fed on a low iodine diet were used.
Method; Rats were divided into two groups, each group was consisted of 5 rats. One group was kept at room temperature of 29°C for at least 2 weeks before experiment, the other was kept also at 23°C for 2 weeks. Then these two groups were exposed to cold environment of 15°C, 8°C, 0°C & -10°C. Blood samples were obtained with heparinized syringes at varying intervals by the puncture of jugular vein under pentobarbital anaesthesia, beginning 30 minuites after cold exposure and immediately centrifuged. The plasma was stored in sterile vials at -20°C until TSH assay. The pituitary glands were collected on ice immediately after decapitation, homogenized with sterile cold saline and centrifuged. These were also stored at -20°C until assay. Thyroidectomy was performed under pentobarbital anaesthesia 2 weeks prior to the experiment. Finally, effect of pretreatment with thyroid hormone, pentobarbital and atropin sulfate on the pituitary thyroidal response to cold were investigated.
TSH assay; Each blood sample was assayed by Mckenzie's modification using DD strain (about 15 gm.) of female mouse.
The results are summarised as follows;
In rats acclimatized to room temperature of 29°C, plasma TSH levels were significantly increased in 30 minuites (P<0.01) after cold exposure to 0°C. On the contrary, rats acclimatized to relatively low room temperature of 23°C showed mild increase of plsma TSH levels in 30 minuites after cold exposure, but this response was not significant. When rats were exposed to a severe cold of -10°C, there was almost no increment of the plasma TSH content in 30 minuites. The speed and the magnitude of TSH response to cold was greatly influenced by the temperature under which the animals were acclimatized prior to cold exposure.
In rats, which were exposed from 29°C to 0°C for 6 hours, plasma TSH levels were increased markedly already as early as 30 minuites as abovementioned. Its peak value was found in 1 hour (P<0.01) and this high TSH level was kept so for 3 hours. (P<0.01) In parallel with this follow up of plasma TSH response to cold, pituitary TSH content wasinvestigated simultaneously. Reduction of TSH content was observed in 30 minuites (P<0.05) and in 1 hour (P<0.05) after cold exposure, then they returned to control level in 3 hours. It is posturated that in an acute phase of thyroid response to cold, TSH release from the pituitary takes place as first step and then TSH synthesis in the pituitary is initiated for supplement of accelerated TSH release in the cold environment.
Thyroidectomized animals showing high plasma TSH levels 2 weeks after operation were exposed to cold at 0°C for 30 minuites. Increase of plasma TSH levels were observed also in this case. It is suggested that TSH response of thyroidectomized animals to cold was rather similar to normal acclimatized animals.
Pretreatment with thyroid hormone (T₃ or T₄) given 2-4 hours prior to cold exposure suppressed pituitary TSH release, so it is suggested that elevation of thyroid hormone may act via the feedback mechanism to decrease TSH secretion.
Among pretreatment with pentobarbital-Na or atropin sulfate, only large dosis of atropin sulfate prevented TSH secretion from the pituitary.
The essential role of the TSH response to cold in connection with thyroidal function was discussed.

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