Recently the method of hormone research has become more precisely and has been complicated. While for routine clinical use, simplicity and rapidity have been required to determine the steroid hormones.
For this purpose Dingman et al. (J. clin. endocrinol., 22 : 87, 1962) have described a simple method for the determination of urinary pregnanediol and pregnanetriol by glassfiber paper chromatography. In our report, too, a more rapid method, that has been obtained from the research of simultaneous extraction of pregnanediol and pregnantriol existed in the same β-glucuronidase-hydrolysed urine and from the scrutiny of solvent system of glass-fiber paper (No. X-934-A.H.) chromatography, is established.
Principal results of our investigations are summarized as follows :
(1) Hydrolysis and extraction.
β-glucuronidase hydrolysis is suitable for the cleavage of urinary pregnanediol and pregnanetriol. Extraction of both steroids is done simultaneously with chloroform and washing with 1.0 N NaOH is adopted. Recovery of pregnane diol is 99.0±0.7% and pregnanetriol is 87.5±6.1%.
(2) Separation on simple glass-fiber paper.
The Rf of pregnanediol and pregnanetriol are sufficiently separated from other steroids in benzene, benzene-isooctane or cyclohexane-acetone system of solvents by glass-fiber paper chromatography. Development is done 20cm from origin (cir. 20min.) at room temperature.
(3) Recovery of glass-fiber chromatography.
EtOAc is employed for extracting after separation of steroids as excellent solvent. It indicates that transferation of large dosis of steroids is not suitable in this procedure.The suitable dosis of transferated steroids are 10-20ug. Recovery of these dosis is about 90%. Absorption curves of contaminants show generally some straight line between 380 and 480mu.
(4) An attempt of colorimetry.
The comparison of colorimetry of steroids at room temperature and 100°C on a water bath are investigated. On pregnanediol the later procedure increases the optical sensitivity but pregnanetriol is adequated at room temperature. Absorption optima of pregnanediol is at 425m.0 and pregnanetriol is at 440mu.
(5) Basing on the results described above, a new rapid method is recommended for estmation of pregnanediol and pregnanetriol in human urine. Then a few data applicated to clinical cases are demonstrated.
Glass-fiber paper chromatography has a consinerable adventages, which are rapidity and accuracy of separation of steroids and simplicity of elution procedure. So these techniques are suitable for routine, simple and rapid determination of steroids.
Besides, recommended method has other beneficial ideas of (a) the introduction of simultaneous hydrolysis of pregnanediol and pregnanetriol with β-glucuronidase, (b) the use of chloroform and 1.0 N NaOH for simultaneous extraction and washing of these steroids and (c) rapid colorimetry of pregnanediol.

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The catecholamine contents in the adrenal gland and adrenal venous blood of rats were measured in order to investigate the effect of vasopressin on the release of catecholamine from the adrenal medulla.
1) Single or repeated intraperitoneal injections of vasopressin caused no significant change in the catecholamine content in the adrenal glands.
2) During the vasopressin infusion, the amout of adrenaline released into the adrenal venous blood increased in 5 out of 11 rats used, while little change was recognized in the other 6 rats. No appreciable change in noradrenaline release was observed by the infusion of vasopressin.
3) The effect of acetylcholine to stimulate the release of adrenaline from the adrenal medulla was exaggerated by the simultaneous infusion of vasopressin, whereas the release of noradrenaline due to acetylcholine remained unchanged.
The author inferred from the present results that there may be some possibility for vasopressin to increase the release of adrenaline from the adrenal medulla, though it failed to exert any significant influence on the catecholamine content of adrenal glands. Further, vasopressin might enhance the sensitivity of the adrenal medulla to acetylcholine.

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It is well known that anti-inflammatory steroids have an inhibitory effect upon the pituitary-adrenocortical function (P-A function). This fact is clinically important, because the patients who have been given these steroids for a long-term period have chronic and subclinical adrenocortical hypofunction. We often observe that they have acute adrenocortical insufficiency when they are exposed to stress.
Furthermore, this inhibitory effect has been widely used in the diagnostic procedure in differentiating adrenal hyperplasia from adrenal tumor (so-called suppression test) or the treatment for adrenogenital syndrome.
Recently, some reports suggest that anabolic steroids may prevent this inhibitory effect upon the P-A function.
The present study describes : (I) the minimal suppressive dese of dexamethasone and the suppression test in the patients with pituitary and/or adrenocortical dysfunction, (2) the adrenocortical reserve in the patients with long-term steroid therapy, and (3) the influence of anabolic steroids on the inhibitory effect of anti-inflammatory steroids.
Exp. 1 Suppression test
Dexamethasone was given by mouth every 6 hours for 4 days. Collections of 24-hour urine were made and total 17-OH-CS was determined by a modification of the method of Reddy, Jenkins and Thorn (1952) or Glenn and Nelson (1953).
The results were as follows :
1. The subjects with normal P-A function (8 cases) underwent no cnange in their P-A function by the administration of 0.25 mg. of dexamethasone per day, but complete suppression occured with a dosage of more than 0.5 mg. per day. It was obvious that in spite of the administration of the same dosage, the day of maximal suppression and escape phenomenon after steroid medication, showed individual variations. In some cases, after the withdrawal of dexamethasone, so-called rebound phenomenon was observed.
2. Two cases of Cushing's syndrome due to adrenal hyperplasia showed no adrenal suppression by the administration 0.5 mg. of dexamethasone per day. When these patients received 6.0 mg. or 2.0 mg. per day, they were showed a complete suppression. In 2 cases of Cushing's syndrome due to adrenal tumors, it was clear that no adrenal suppression was observed by the administration of 6.0 mg. of dexamethasone per day.
3. In hyperthyroidism (16 cases) slight or moderate suppression was observed even by the dosage of 0.25 mg. per day.
4. Two cases of acromegaly (one with diabetes mellitus) showed no suppression by the dosage of 0.5 mg. per day. In one case, after radiation therapy with ⁶⁰Co of 6000 r in total dose, no adrenal suppression was observed by the dosage of 0.5 mg. per day.
Exp. 2. Arenocortical reserves in the steroid-treated patients.
So-called adrenocortical reserves were estimated by use of ACTH stimulation. 40 I.U. of ACTH-Z was given intramuscularly at 6.00 a.m. for 3 days.
The results were as follows :
1. In 15 cases ACTH stimulation was performed within 5 days after the cessation of steroid therapy. Urinary output of total 17-OH-CS following ACTH stimulation in the patients treated with anti-inflammatory steroids were significantly smaller than control subjects with normal P-A function. A significant negative correlation was found between their adrenocortical reserves and the logarithm of duration of steroid treatment or the logaritnm of total doses of administered steroids. There was no significant correlation between their adrenocortical reserves and mean values of daily dosage or ba se line 17-OH-CS before ACTH stimulation.
2. Adrenocortical reserves of 5 patients who had been treated with anti-inflammatory steroids were determined a few months aftes the cessation of steroid theraphy.Three patients who had been treated for 10 days to 30 days showed normal reserves. However, 2 patients who had been treated for 210 or 448 days showed subnormal or decreased reserves.

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In order to elucidate the role of autonomic nervous system on the regulation of parathyroid function, the oxalate tolerance test was performed in normal, thyroparathyroidectomized, vagtomized and atropinized dogs. Plasma or serum samples were obtained to determine the total calcium content before, immediately after and up to 6 hours following a 30 minutes infusion of 40 mg/kg 0.4% sodium oxalate solution in physiological saline. The recovery was significantly faster in dogs bilaterally vagotomized than in intact animals. Similarly accelerated recovery was also observed in animals in which one of the 3 groups of original rootlets of the accessory nerve (AH rootlets of Okinaka), which later joins the vagus, was bilaterally sectioned intracranially immediately after their departure from the medulla oblongata. Administration of atropine during the test period also resulted in an accelerated recovery. In thyroparathyroidectomized, thyroparathyroidectomized-vagotomized and thyroparathyroidectomized-atropinized dogs the recovery was apparently retarded. Acceleration of the recovery from hypocalcemia approximately paralleled the dose of parathyroid hormone in thyroparathyroidectomized dogs.

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Since the discovery of insulin in 1922, much progress has been made in the therapy for diabetes mellitus. However, insulin has its limitations in that it can be used only by means of injections.
For a long time, we have been looking forward to the discovery of a per oral medicine which would be applicable. Recently, various per oral medicines have been discovered : mesoxan, sulfonylurea and biguanide. However, there are divergent opinions among the researchers throughout the world regarding the mechamism of hypoglycemic action of these medicines.
At the Congress of the Japanese Society of Internal Medicine, Professor Hideo Yoshida of our Medical Department reported on the mechanism of sulfonylurea. (1958) At the Congress of the Japanese Endocrinological Society, Professor Hideo Yoshida et al. reported on the mechanism of biguanide. (1961)
My present study not only covers the influence of these per oral medicines on carbohydrate metabobolism, especially pyruvic acid and lactic acid, but also on the effect of glucose tolerance test.

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Aldosterone excretion in urine and salt concentration in serum were studied on 4 subjects on different intakes of sodium chloride.
Results obtained were as follows ;
1) Daily output of aldosterone in urine increase remarkably on low sodium diet and decrease on high sodium diet. The other corticoid, such as, 17-OHCS and 17-KS is maintained constant irrespective of salt intake.
2) An inverse correlation is found between the aldosterone output in urine and the logarithm of dose of Na content in urine. The K/Na ratio bear a good positive correlation with the aldosterone output. Remarkable individual difference were found in the correlationships.
3) Na and Cl concentration in serum increase on high sodium diet and decrease on low sodium diet, while K concentration in serum is maintained constant.
4) The change of Na concentration in serum seems to bear important factor which governs aldosterone excretion.

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