Variation of output of urinary estrogens before and after oophorectomy or adrenalectomy, or both, in patients with uterine cancer, recurrent or metastatic breast cancer, have been observed by using modified Ittrich's chemical method of estimation. Concurrently, the following have also been investigated :
1. The effects of administration of ACTH, PMS, HCG, corticoids and synthesized gestagen (norethindrone), on the excretion of estrone, estradiol-17β, and estriol in urine.
2. The problems of estrogen metabolism in human liver, in patients with breast cancer, after castration, right adrenalectomy and anastomosis of the left adrenal vein with renal vein, and of secretion of estrogens from the ovaries in postmenopausal women after adrenalectomy.
3. The effects of subtotal adrenalectomy against Cushing's disease on the symptoms and estrogen milieu.
The following results have been obtained :
1) It was found that there is an abrupt decrease in the output of urinary estrogens soon after oophorectomy, and that the recovery of urinary estrogen level noted after a while, and stabilized due mainly to recovered estriol value. As a result of a decrease in estradiol-estrone fractions and relative increase in estriol fraction, higher quotient of ET/EO+ED was observed after oophorectomy.
2) Marked increase in the output of urinary estrogens, particularly of estriol fraction was observed when ACTH was administered to castrated women with intact adrenals. No apparent response to the administration of gonadotropin after oophorectomy was obtained.
3) Marked decrease in the output of urinary estrogens, particularly of estriol fraction when prednisolone and dexamethasone was administered to castrated women.
4) Decrease in amount of estriol in urine when small doses of norethindrone were administered to castrated women.
Increase in the output of urinary estrogens, mostly of estrone fraction, followed by an increase in estradiol fraction when large doses of norethindrone were administered to castrated women or castrated combined with adrenalectomy.
It is assumed that this is due to extraglandular conversion of 19 norsteroids, particularly to estrone first and then estradiol-17β.
5) Further reduction in the output of urinary estrogens (mean value, 3.7μg/day) have been observed and no response was seen with ACTH after oophorectomy and adrenalectomy.
6) On the other hand, a slight increase in the output of urinary estrogens has been observed when ACTH was administered to castrated women with right adrenalectomy and anastomosis of the left adrenal vein with renal vein. This is probably due to a gradual process of the estrogen molecule disintegration in human liver and an overflow-phenomenon beyond its capacity.
7) There was a maintenance of excretion of small amounts of estrogens in urine. Still secreted from the ovaries in postmenopausal women after adrenalectomy.

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The thyroid stimulating hormone TSH is one of the proteohormones secreted from the anterior lobe of the pituitary. TSH retaining active biological property of proteohormone, however, has not been fully known and as yet it has to be further purified. The true nature of TSH can be ascertained only when successfully purified to a minimum unit.
The present investigation is an attempt made on purifying TSH by means of ion exchange cellulose column chromatography by taking a crude TSH active substance Pretiron (Schering), and studying the relationship between TSH activity and carbohydrate content of TSH.
1. Purification of TSH by ion exchange cellulose column chromatography
For ion exchange cellulose, CM-cellulose and DEAE-cellulose were prepared from powdered cellulose available, and batch technique combined with chromatography was tried. For an optimum condition of purifying TSH, a column of DEAE cellulose was best used by dissolving with tris-buffer at pH 7.6. With this purification method, it was possible to obtain 60 jsu/mg of TSH active substances of which a unit was found to be twice as high as the original Pretiron. This fraction was examined by paper electrophoretically under various pH buffers and we succeeded in isolating a fraction which would present a single pattern at any pH. This supposedly paper electrophoretically identified purified fraction has shown not only content of 60 jsu/mg TSH active substance but it was also some minute quantity of gonadotropic active substance. Although it is possible to obtain and isolate a higher active unit per weight of purified TSH by means of DEAE-cellulose column chromatography, there still remains a further problem of separation of gonadotropic active substance which was dissolved in a fraction near ion concentration 0.4. However, a higher TSH active substance could be obtained in the fraction in the vicinity of 1.4.
2. Purification of TSH and its carbohydrate content, br>Purified TSH active substance was initially obtained through DEAE-cellulose column chromatography and with continuous paperelectrophoresis. The relationship between biological activity and its carbohydrate content was studied. Quantitative measurement of hexose, and hexosamine and sialic acid were made. Hexose was measured by the method of orcinolsulfuric acid using galactose as a standard substance. Hexosamine was determined by the modified Elson-Morgan method using d-glucosamine hydrochloride as the standard substance. Estimation of sialic acid level was done by thiobarbituric acid method, using N-acetyl neuraminic acid originally devised by Yamamoto of Teikoku Hormone Manufacturing Company. For TSH activity Kishimoto's modified Bates method was applied.
(a) Carbohydrate content in purified TSH, with cellulose column chromatography
Pretiron was examined for TSH active substance and its carbohydrate content, and the fraction was extracted by DEAE cellulose column chromatography. It was found that purified TSH active fraction contained a higher unit of hexose than that of unpurified Pretiron, while an amount of hexose increased in direct proportion to further purification of TSH. It was revealed that a large amount of hexosamine was found in low TSH active fraction but hardly any was found in a higher active substance demonstrating the fact that hexosamine is unrelated with TSH. Sialic acid was found to be low compared with Pretiron in which TSH activity was less purified. When the TSH substance was low hardly any sialic acid could be found, though hexose and hexosamine could be detected. It seems that sialic acid is concerned with TSH in some respect.
(b) Carbohydrate content in purified TSH, with continuous paperelectrophoresis.
It was found that the quantity of hexose increased parallel with increased purification of TSH.
3. Variation of TSH activity and carbohydrate content with reference to pH.

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Recent extensive studies of oral antidiabetic agents, particularily two kinds of hypoglycemics, sulfonyl ureas and biguanides, greatly attracted our attention because of its high degree of hypoglycemic activity and low toxicity. But these compounds have not been yet satisfactorily recommended as a perfect antidiabetic drug. There remains further elucidation of the mechanism of the hypoglycemic action. The author's purpose of the present studies is to obtain some more information about the hypoglycemic action of certain natural organic substances, amino acids, chlorpropamide as a sulfonyl urea derivative and metformin as a biguanide derivative.
The results reported were established in normal male and female, non-pregnant rats (Wistar strain), weighing over 100 g. All blood sugar determinations were performed in a fasting state, according to method of Somogyi and Nelson's blood sugar determination.
1) The following natural organic substances and amino acids were orally administered in normal rats, with doses of 100 mg/kg of cholic acid, 50 mg/kg of desoxycholic acid, 200 mg/kg of hippuric acid, 200 mg/kg of theobromine, 50 mg/kg of caffeine, 100 mg/kg of theophylline, 700 mg/kg of DL-norleucine, 200 mg/kg of L-cystine, 500 mg/kg of tyrosine and 700 mg/kg of DL-creatinine.
a) Although cholic acid, desoxycholic acid, caffeine, theophylline and tyrosine disclosed slight hypoglycemic action, a potent prolonged activity of blood sugar lowering effect was recognized in theobromine. Its maximal hypoglycemic reduction ratio of blood sugar level showed 38.6 per cent in 5 hours after the administration and the duration of hypoglycemic action over 6 hours.
b) No hypoglycemic effect was observed in hippuric acid, DL-norleucine, L-cys-tine and DL-creatinine.
2) In the mean number of β-cell count of the Langerhans' islet of the alloxan diabetic rats, treated orally with doses of 200 mg/kg of theobromine in the 7 to 60 day's duration of diabetic state, the degree of 14.1 counts and more of β-cell per a islet (more than approximately 1/5 of β-cells in a normal islet) revealed relatively marked lowering effect. On the other hand, that of 10.2 counts and less of β-cells per islet (less than 1/6 of β-cells in a normal islet) was not active in hypoglycemic effect.
3) The oral administration of theobromine in doses of 200 mg/kg in normal rats for a month showed no histologic change, including Langerhans' islet, except only a mild cardiac hypertrophy.
4) The intravenous administration of 300 mg/kg of chlorpropamide in normal rats lowered the blood sugar level in 55.5 per cent at maximum (the post-administrative 4 hours-value on the average of 5 cases) and its action was prolonged for 5 hours and more in duration. In addition, the intravenous administration of metformin, 200 mg/kg, revealed the blood sugar lowering effect in 62.8 per cent at maximum (the post-administrative 2 hours-value on the mean of 5 cases) and maintained for 5 hours and more in its effect.
5) In the experiments of the oral administration of theobromine in doses of 200 mg/kg, the intravenous administration of chlorpropamide in doses of 300 mg/kg and metformin in doses of 200 mg/kg in normal rats, the blood sugar levels were measured under our interruptive treatment of blood supply in the pancreas, and the alteration of β-granules in Langerhans' islets was observed after the sacrifice at each hypoglycemic condition. The results obtained were as follows :
a) Theobromine caused the degranulation by its direct action on β-cells of Langerhans' islets of the pancreas, resulting in its hypoglycemic activity through the release of insulin. b) Chlorpropamide lowered the blood sugar in the same mechanism as theobromine. c) Metformin, on the contrary, showed its hypoglycemic action, both mainly by extrapancreatic mechanism and partly by pancreatic mechanism, namely by the secretion of insulin.

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