Paper electrophoresis studies were made on the serum protein fractions in rabbits given powdered thyroid gland (30 mg/kg every day, per os) or undergoing thyroidectomy, and in the offspring of rabbits (F₁, F₄, F₅ ; these were not given powdered thyroid gland nor did they undergo thyroidectomy) descended from ancestors given powdered thyroid gland (30 mg/kg every day for one month, per os) or undergoing thyroidectomy (more than one month after operation) over several successive generations. Compared with normal values, the results were as follows.
1) Average values in 8 normal rabbit sera (4 female, 4 male) were ; albumin 60.7±1.7%, a-globulin 13.0 ±2.0%, β-globulin 12.2 ±2.1% and γ-globulin 14.1±1.9%. Total protein values were 6.39±0.24g/d1.
2) Alterations presented with the passage of time after the administration of powdered thyroid gland in normal rabbits were, albumin was markedly decreased, a-globulin and γ-globulin increased after 10 days and after that continued similar changes. Changes after thyroidectomy schowed gradually a decrease in albumin and an increase in β-globulin, and these findings became more evident as time passed.
3) Serum protein fractions in offspring of rabbits (F₁, F₄, F₅) administered powdered thyroid gland over successive generations showed a decrease in albumin and an increase in a-globulin, and these changes seemed more and more evident as the generations progressed, and resembled the values in rabbits given powdered thyroid gland.
4) Serum protein fractions in offspring of rabbits (F₁, F₄) undergoing thyroidectomy over successive generations showed a decrease in albumin, a slight increase in α-globulin and an increase in β-globulin. These changes also seemed more evident as the generation progressed, and resembled the values of rabbits after thyroidectomy.

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Studing on biological actions of TSH employing Pretiron (A.G. Schering) as this source, we found that the administration of this preparation has induced the excessive ovarian responses in mature female rabbits, such as multiple ovulations and corpora lutea formation-Friedman's Reaction.
The data suggests that it has a relatively high gonadotropic potency or surely it is contaminated with gonadotropic hormones. So we tried to estimate the gonadotropic potency of this preparation and, moreover desirably, to separate gonadotropic substances from this.
We used both mouse uterine weight test and Junkmann-Schoeller's method for gonadotropin and for TSH estimation respectively.
It has found that Pretiron had relatively high gonadotropic potency ; 10-J.S. unit of it showed at least I m.u.u. of gonadotropic activity.
Then, in order to separate gonadotropic fraction from TSH preparation, we applied Condliffe's column-chromatography method, employing IRC-50 as adsorbent.
We used 0.2M-Na-phosphate-buffer solution for the first elution, and for the second 1.0-M-NaCl-solution. The former fraction we named Fraction-I, the later Fraction-II.
The biological assay of Fraction-I appeared to be mostly specific for the gonadotropic hormone, but for the thyrotropic activity we could recognize nothing in it.
On the other hand Fraction-II showed biologically high thyrotropic activity but not any gonadotropic potencies.
For these reasons we can say that this column-chromatography method is very usefull to separate gonadotropic substance from TSH Preparation, namely to purify the TSH preparation for biological applications.

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Choosing albino rats of the Wistar strain for study, the author destructed the thyroid glands of the rats of both sexes using radioactive iodine I¹³¹, crossed them above 1 month later and made them sire litters (F₁). The destruction of thyroids of the young, both male and female, was achieved 3 months after birth in a similar manner that their parents were treated, mated above 1 month post-administration and made to give birth to the F₂ offsprings. The same procedure was conducted on four generations till the fourth descendants were obtained thereby, scheming to explore the influence of radiothyroidectomized state upon the descendants of successive four generations. On the offspring rats F₁ (26 cases), F₂ (26 cases), F₃ (29 cases) and F₄ (40 cases), each generation was subjected to the study of growth rate by determining body weights, to the comparative study of the weights of splanchnic organs at a definite time post partum, to the histological and histometrical investigations of thyroids and other splanchnic organs and was rendered to the measurements of their serum PBI, of serum total cholesterol and of the rate of thyroid uptake of I¹³¹ and to the comparison with those of the control animals (25 cases), yielding the following results :
(1) From the results of the measurement of body weights, it was revealed that the gain in body weights as compared to those of controls was reduced by degrees as the generation went down.
(2) The ratio of thyroid-to body weights tended to increase as the generation descended.
(3) In the picture of thyroid glands, follicles grew larger on and on, follicular cavities were filled with colloids, became to take up much eosin and PAS dye and the formation of peripheral vacuolation were depressed as the descent of generation went on.
Follicle cells went on flattening, PAS granules seen in follicle cells were decreased in number parallel to the descent of generation, and the majority of follicle cells were found to present a similar appearance to that of endothelial cells.
(4) From the histometrical examination of thyroids, the following results were obtained a) Follicle areas measured 5012±524μ² in controls, 5645±539μ² in F₁, 6539±846μ² in F₂, 7048±863μ² in F₃ and 7513±1048μ² in F₄, tending to grow larger as the generation descended.
b) Areas of follicle cavity were of the dimension of 2468±498 in controls, 3527±654μ² in F₁, 4457±526μ² in F₂, 5403±322μ² in F₃ and 6086±676μ² in F₄ which shows that the value of controls was about one half that of F₄.
c) Areas of follicle cells ranged to 129.3±16.7μ² in controls, 108.6±25.6μ² in F₁, 96.1±21.2μ² in F₂, 75.9±12.4μ² in F₃ and 66.0±11.7μ² in F₄, which means the fall on successive generations.
d) Follicle cells were of the height of 7.7±0.585μ in controls, 6.8±0.937μ in F₁, 6.1±0.789μ in F₂, 5.3±0.603μ in F₃ and 4.4±0.44μ in F₄, showing a tendency to an apparent decrease on consecutive descent of generations.
e) Cell counts per follicle cell amounted 19.9±2.53 in controls, 20.3±2.41 in F₁, 20.3±3.96 in F₂, 21.6±2.26 in F₃ and 20.7±2.53 in F₄, minor changes being observed in cell counts.

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A new technique for the quantitative determination of TSH, based on the measurement of the hormone in stimulating thyroidal uptake and release of inorganic radioiodine, was further investigated using surviving bovine thyroid slices in vitro. The results were as follows :
1) TSH, added in vitro, stimulated the uptake and decreased the release of inorganic iodine by the slices.
2) According to the results of the chromatographic separation, the radioactivity discharged into the medium after the release experiment was not of hormonal radioiodine but of inorganic.
3) In the uptake experiment, when TSH and radioiodine were added simultaneously, no usefull TSH dose-response proportionality was obtained. There were statistically significant differences in the radioiodine uptake among 10^-4, 10^-3, 10^-1 and 10-1 JSu/ml of TSH when the thyroid slices were incubated with TSH for 4 hours prior to the addition of radioiodine. However the reproductivity of these relationships was very poor.
4) In the release experiment, statistically significant linearity and differences were observed over the range from 10^-5 to 10^-2 JSu/ml of TSH, but there was a great variation in the TSH dose-response curve and the mean λ value was 0.68, being higher than that obtained by any other recent techniques.
5) For the purpose to increase the slope of the dose-response curve and augment the precision in the release experiment developed by Bakke et al., the author has made some improvements on their method, and obtained satisfactory outcomes for the most part. This procedure seems to offer a fairly convenient and sensitive technique for the assay of TSH ; with a mean λ of 0.34, as precise as that obtained by current methods.
6) 1 USP unit was estimated to be equivallent approximately to 10 JS units by the improved release experiment applied to the 4 or 6 point method.
A modified assay technique for thyrothrophin (TSH) based on the release of inorganic radioiodine by bovine thyroid tissue slices in vitro was designed for its clinical application and was used in titrations of serum TSH levels in various disorders.
In normal adults, the mean serum TSH level was 1.82±1.38 (standard deviation) milli JSU/ml in 15 males in the 2nd-4th decades, 0.89±0.54 milli JSU/ml in 5 males above the 5th decade, and 1.91±1.49 milli JSU/ml in 21 femeles in the 2nd and 3rd decades. The confidence limit (P=0.05) of serum TSH level in 41 normal adults was 2.2-1.3 milli JSU/ml, and its seasonal changes was estimated.
In simple goiters, the serum TSH level in PAS induced-and endemic diffuse goiter was rather high, but in cystic or nodular type it was normal or partially low. In these euthyroid patients, a negative correlation between the TSH level and the PBI value in serum was observed.
In primary hypothyroidism, the serum TSH level was markedly elevated. On the contrary, in secondary hypothyroidism it was extraordinarily decreased or beyond measure.
In hyperthyroid patients, the serum TSH level varied from less than 0.1 to more than10 milli JSU/ml, its mean value was in upper part of the normal range, and abnormally high or low levels of TSH in serum were observed not only in the group with exophthalmos but also in the group without exophthalmos, whereas the former showed somewhat high level of TSH in serum on the average. Two euthyroid patients without goiter but with marked exophthalmos did not differ from normals in their serum TSH levels. In the group of hyperthyroidism, in which the thyroidal radioiodine uptake curve showed the “descending type”, exophthalmos was more frequenthy observed and the serum TSH level tended to be higher than that of the group showing the “ascending type” of the thyroidal radioiodine uptake curve.

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To investigate relationships between fine structure and function of the thyroid, morphological changes of follicular epitherial and interstitial cells of the gland produced by administration of I¹³¹ was examined with the electron microscope.
The male rats fed on low iodine diet were used to which 300μc of I¹³¹ was administered by a single intraperitoneal injection of NaI¹³¹. One, 12 and 24 hours, 4, 9 and 30 days after I¹³¹ administration, the glands were removed and fixed in isotonic 1% OsO₄ for ultrathin-sections. The results obtained are summarized as follows.
1. At one hour after I¹³¹ administration hardly any changes in fine structure of epitherial and inerstitial cells were observed.
2. At the earlier period after I¹³¹ administration (4-9 days), height of epitherial cells or of their microvilli decreased, nuclear materials aggregated and the appearances of cytoplasm became remarkably denser.
3. At this stage, mitochondria swelled, vacuolization and disarrangement of their cristae being observed, while Golgi complex were scarecely observable and electron dense granules of round or irregular shape (probably lipid granules ?) increased.
4. The profiles of endoplasmic reticulum changed remarkably day after day. At 12 and 24 hours after I¹³¹ administration, saccate and polymorphic shape of endoplasmic reticulum diminished and, after 4 and 9 days, lamellic and small vesicular shapes were observed only in cytoplasm.
5. But 30 after I¹³² treatment, the follicular cells as well as their microvilli recovered their normal heights, the nuclei also became almost similar to the normal one and Golgi complex reappeared. Cytoplasm gave a foam-like appearances because of the increasing of saccate and polymorphic shape of endoplasmic reticulum.
6. Morphological changes in interstitial cells in the thyroid were in general trend, similar to those of the follicular cells.¹³¹From the above findings it might be said that changes in the fine structure of the thyroid gland at the earlier period are related to the hypo-functional state of the gland produced by internal irradiation due to I¹³¹ and relationships between secretory ability and the fine structure of endoplasmic reticulum are discussed.

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Rabbits, rats and guinea pigs were singly administered with alloxan 200 mg/kg, dithizone 200 mg/kg and oxine 50-100 mg/kg respectively ; the blood sugar volue is measured 1.5, 3, 5, 8 10, 17, 24, 48 hours respectively after the injections. The animals were constantly of hyperglycemia by means of the chemical administration. Despite the effect of the chemicals was variably in degree with the kinds of animal, it was common to all animals that the temporary initial hyperglycemia is followed by the hypoglycemic status in 1-3 hours, and the severe hyperglycemia is successive.
As the control to the chemical injection, another rabbits were administered with 40% hypertonic solution of glucose, 20 cc/kg/day for 7 days. The histological pictures of the islets were examined 24 hours after the injection ; according to the preparations of chromalume hematoxylin phloxin (CHP) stain, a majority of cells in islets appeared to be positive to phloxin (P) with the consequent impossibility for the identification of alpha-and beta-cells. In the latter, CH-positive granules or filamentous bodies decreased in number or almost disappeared. It was frequently encountered that the P-positive cylinderic elongated cells were arranged radially, as if it were a petal facing the capillary lumina, and the connective tissues concurrently grew in the adherences of the blood capillaries.
In the temporary initial phase of hyperglycemia in rabbits administered with alloxan, CH-positive bodies did not so much decrease as by glucose administration. In spite of the expectation for the integral multiplication of them in the beta-cells in the hypoglycemia, they were, in fact, absent in 7 hours, which may deal with the releasing pattern from the cell-bodies gaining the subsequent strong affinity of P ; in 48 hours all of the cellular consistuents of islets were diffusively P-positive, in which were sometimes visible the aforementioned petal-like arrangement surrounding the closing lumina of the capillaries. Because of the comprehensive disappearance of CH-positive bodies, there were no longer the islets containing the typical beta-cells. Notwithstanding the profound hyperglycemia, any signs of degeneration in islet cells were not affirmative, at least the vanishment of the cellular elements following the necrosis and collapse or disintegration did not arise in the present observation. The histological changes in islets of rats with alloxan administration was same with those in rabbits. In particular, even when the animals were in the hyperglycemia in 48 hours, a small amount of CH-positive bodies were conservated in most of beta-cells, showing the fact that rats were not so much influenced by alloxan administration as rabbits. The alpha-cells accumulated in the peripheral layer of islet forming the syncytium became to have the apparent contour and to invade the internal areas of it by growing of cell population. The changes in guinea pigs corresponded in general to those in rabbits, where the pattern of the degeneration and collapse was also not visualized.
Dithizone was capable to permit rabbits to fall into glycemia, in spite of the less effectiveness to the other animals. Morphological aspect of this compound to the islet cells resembled those of alloxan. However, it was a common observation that in the initial hyperglycemia in rabbits most of cells fall into the abnormal and augmented hyperplasia, whose cytoplasm was dense homogenous P-positive character on account of the releasing-out of CH-positive bodies. In the highest level of hyperglycemia caused after 48 hours, however, the patterns showing the considerable restoration from the inhibition of cell activity were present in a fashion that P-negative cells containing the numerous CH-positive bodies are commenced to take places in a certain locations of islets again. Of course, there were no signs involving in a degeneration or collapse in islets.

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The mode of hormonal regulation of adrenal corticoids to sweat glands in plantar skin of rats were investigated on the cytological observations. With the materials of 35 rats of Wistar strain weighing 130-150 g as well as 5 rats weighing 280 g, following experiments were undertaken to determine this problem, as normal untreated control, bilateral adrenalectomy, prolonged subcutaneous administration of 2.5 mg of DCA per rat for 20 days after the removal, contineous subcutaneous administration of 2.5 mg of cortisone for 20 days after the deprivation, and subcutaneous injection of 0.4% formalin for 5 days to the normal untreated as the agencies of the chronic stressor. It is characteristic that this approach was carried with the same animals from whom the various samples of plantar skin were successively withdrawn during the stages of the experiment.
The terminal portion of the sweat glands of the normal male rats consists of two types of the glandular cells, a pale and dark cell, which manifest the irregular and complicated contours, and sometimes provide the obsecure outline especially in a pale cell, therefore it was not always afforded that they are distinguished from one another in their distribution in the epithelium. The intermediate type between both cells were occasionally recognized, showing their mutual transformation. At the basal part of the cell-body of the dark cell filamentous fibrous structure, larger and longer than the mitochondria, were situated perpendicularly, and in particular accumulated pronouncedly in the bifuricated foot-like basal cytoplasmic protrusions strongly stained. Several number of mitochondria, however, appeared in various parts of cytoplasm of most of cells, whereas the large secretory granules were almost negligible in them. A small amount of glycogen appeared around the nucleus as a dot in many of the cells.
Five days after the adrenalectomy, the pale cells increased in number and the fibrous structure in them became vague. They resembled so-called basal cell in their distribution and shape. It was noted that the dark cells are apt to take a position of the surfacing cells within the epithelium. These change became gradually prominent in the advancing days after the adrenalectomy. Within 20 days the latter cells were multiplied and provided with the obvious bifurication of the foot-like protrusions connected to the basement membrane, whereas the pale cells included the numerous vacuoles which may be a sign of the degeneration. The glycogen content increased in both cells.
In the prolonged administration of DCA, the aforementioned alterations were considerably recovered, nevertheless the restoration is not thorough so much as the normal level. From the appearance of glycogen content, it was established that DCA may be a powerful stimulator to permit them to recover from the probable hypoactivity induced by the ablation of adrenal gland.
A single administration of cortisone to the adrenalectomized rats did not make the pattern of restitution from the adrenalectomy, while the prolonged injection for 5 days and over enabled them to recover sufficiently. Notwithstanding this phenomenon, cortisone is inferior in the ability of promoting the cell function to DCA. The persistent injection of cortisone for 15-20 days made some degenerations in most of the cells, when the glycogen concentration did not go in parallel with the degree of the degeneration, because the amount of glycogen was less than that by the adrenalectomy.
The administration of cortisone to the normal, rats did not induce a sign of the degeneration in the sweat gland cells. However, this may not mean that the hormone is not effective to the normal gland cells, since there is enough reasons that the cells become transparent in general, and simltaneously the fibrous structure is obsecure at the cell basement. The glycogen content increased, in spite of the insufficient recovery up to the adrenalectomy level.

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For the clinical applications of serum protein bound iodine, the method for its determation has to be more simplified and the data should be highly reliable. Along this line, the author contrived a new method applicated the distillation method.
The gaseous iodine produced by reduction was gathered directly into the solution (pH 8.3) of arsenious acid with NaHCO₃. The celium solution was added to this solution which was acidified by H₂SO₄ for producing Ce-As system, thus the serum protein-bound iodine would be estimated by its discoloration.
At first, iodine in the blood specimen was estimated from the standard curve previously prepared, secondary, the blank values were determined as PBI values, hereafter the serum protein-bound iodine estimation was made. Results of the recovery test for this new method distributed within the ranges from 88 to 107 per cent (avarage 95 per cent). The parallel tests showed their differences from 0.2 to 0.77 per 100ml. According to these results we may conclude the date obtained by this method has high accuracy.
The normal level obtained by this method was 4.5-8.0γ per 100ml. (avarage 6.2γ per 100ml.).
The datas in patients of various diseases were obtained as follows :
Pulmonary tuberculosis : 5.5 to 13.0γ per 100ml.
hyperthyroidism and thyrotoxicosis : 8.2 to 19.3γ per 100ml.
tumor of the pituitary gland : 7.0 to 9.5γ per 100ml.
diabetes mellitus : 3. 7 to 9. 6γ per 100ml.
Struma Simplex : 5.2 to 7.3γ per 100ml.
obesity : 2.5 to 6.8γ per 100ml.
hypothyroidism (with incomplete clinical symptoms) : 1.9 to 3.5γ per 100ml.
myxoedema : 1.8 to 2.8γ per 100ml.
Simmonds' Disease : 3.6 to 4.7γ per 100ml.
Nephrosis : 3.5 to 5.6γ per 100ml. Conclusively, the author introduced a new simplified method of serum protein-bound iodine determination and reviewed the datas estimated by this method.

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Alkaline RNA-phosphatase activity was histochemically studied in the pancreatic islet of Langerhans under the influence of hypophysis-adrenal hormones and its defficiency.
Results obtained were as follows.
a) Adrenalectomy caused moderate increase of enzymatic activity which considered as hyperfunction in both cells of islet, especially in alpha cell 7th day after hypophysectomy, but time elapsed to 15th day the degree of the enzymatic reaction of both cells was declined markedly mainly in the alpha cell. ACTH administration after adrenalectomy occurred no remarkable changes in the distribution and intensity of alkaline RNA-phosphatase in the pancreatic islet.
b) Both cells of islet were affected prominently by the hypophysectomy and especially alpha cell showed marked decrease of the enzymatic activity accompanied with the shrinkage of both cells.
c) By the ACTH administration were occured the hypertrophy of both cells and increase of intensity of alkaline RNA-phosphatase in alpha cell which presumed hyperfunction of the cell. Cortirone administration and GH administration also caused similar results.
d) When ACTH was administered to the hypophysectomized-adrenalectomized rat, activity of RNA-phosphatase was as like as in the pancreatic islet of the hypophysectomized-adrenalectomized case.

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