The object of this study was to obtain pregnane-3 (α), 20α-diol in crystalline form which is indispensable as a standard in the quantitative investigation of gregnanediol.
Pregnancy urine (6th. to 8th. month) was hydrolysed with HCl or β-glucuronidase and extracted with toluene. The extract was washed with n/10 NaOH and water, and saponified with 4% NaOH in methanol. The filtrate obtained after passing through a glass filter was evaporated to 5 to 7cc. under reduced pressure. Column chromatographic analysis of this extract was carried out with 5g. of alumina, standardized according to Brockmann. The column was regulated to 0.8 x 10 cm.. As eluant different concentrations of methanol in benzene were used progressively from 0.1 to 10% following development with the 20 cc. of benzene. Crystalline deposits were obtained in 0.1-0.2% and 0.3-0.5% fractions, using concentrations under 1% methanol in benzene. Fractionation of these two crude substances with Girard T reagent showed that 86.6% of the former was a ketonic substance and 95.6% of the latter was non ketonic. I, therefore, presumed that the requisite substance for my study existed in the 0.3-0.5% methanol in benzene fraction.
The crude substance in the 0.3-0.5% fraction was precipitated in 4 : 16 ethanol and water, and then recrystallized in absolute methanol. The obtained crystals were white coloured and hexagonal columnor needle-shaped. The absorption spectrums of H₂SO₄ chromogen of these crystalline substances exhibited their peak at 425 mp and the bottom at 385 mμ to 390 mμ. The absorption spectrums of their H₂SO₄ chromogen incubated at 25°C for 2 hours and 24 hours, according to the method of Zaffaroni, were almost similar ; both of them exhibited their peak at 425 mμ and the bottom at 386 mμ in the visible region, the peak at 330 mμ in the ultra violet region, which was higher than the peak in the visible region. Their melting points showed 234.8°C to 238.4°C with the microscopic apparatus (Hasegawa). As the absorption band did not appear in the region of the infra-red spectrum of these crystalline substances, I found out that they were not ketonic, or that they contained no ketonic substance.
Considering the above investigations I concluded that these recrystallized substances from the 0.3-0.5 % methanol in benzene fraction isolated by my chromatographic technique could unquestionably be recognized as mearly pure pregnane-3 (α), 20α-diol and could possibly be used as standard substances in pregnanediol studies.

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Some of the basic problems in the quantitative analysis of pregnanediol, using pregnanediol obtained from pregnancy urine accoding to our extraction and isotation procedures, were carried out.
1. H₂SO₄ chromogen of pregnanediol : The absorption curves of isolated substances obtained from pregnancy urine (5 liters of each), which had been dissolved in 5.0 ml. of 95% H₂SO₄ and left at room temperature for 30 minutes, were compared. All of the spectrums of the specimens exhibited peaks at 300 mμ. and 420 mμ. and the bottom at 390 mμ.. We, therefore, found out that all of our extracted substances were identical.
2. Relationship between concentration of H₂SO₄ and optical density of H₂SO₄ chromogen of pregnanediol : The optical density of H₂SO₄ chromogen of pregnanediol was elevated as the concentration of H₂SO₄ was increased in both the following conditions ; when 32.4 μg. of the material was left at room temperature for 20 minutes in 5.0 ml. of 65%, 95% and 95% H₂SO₄ and when 21.6 μg. was incubated at 100°C for 20 minutes in 5.0 ml. of each of the above percentages of H₂SO₄.
3. Relationship between duration of incubation and optical density of H₂SO₄ chromogen of pregnanediol : 32.4 μg. of the material was incubated at 100°C in 5.0 ml. of each of the above mentioned percentages of H₂SO₄. The optical density was elevated as the concentration increased for incubation of less than 20 minutes duration, but the optical density became constant as the duration was prolonged for more than 20 minutes.
4. Relationship between lapse after colour development and optical density : The optical density of H₂SO₄ chromogen, when 27 μg. of the material in 5.0 ml. of 95% H₂SO₄ was not incubated, gradually increased after colour development, and became constant after 30 minutes for 30 minutes. When the material was incubated for 20 minutes at 100°C and then was cooled, the optical density became constant just after cooling for 60 minutes.
5. Relationship between duration of incubation and amount of material : When different amounts from 10.8 μg. to 43.2 μg of the material were incubated at 100°C in 5.0 ml. of 95% H₂SO₂, the optical density of each material remained constant after incubation for 20 minutes. The absorption curves of 27 μg. of the material incubated for 5, 10 and 20 minutes were different from that of substances not incubated in the region under 390 mμ., but were not markedly different in the region above 390 mμ.. According to the calibration curve between the above amounts of material and their H₂SO₄ chromogen, we found that pregnanediol was quantitatively estimated in much smaller amounts after incubation for 20 minutes at 100°C than after no incubation.

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The object of this study was to obtain pregnane-3α, 17α, 20α-triol in crystalline form which is indispensable as a standard in the quantitative investigation of pregnanetriol.
The urine of pregnant women (6 th. and 9 th. month) and a normal man who were given 65-125mg of 17α-hydroxyprogesterone capronate (Proluton-Depot : Schering) were hydrolysed with β-glucuronidase, extracted with toluene and chromatographed with 5g. of alumina, according to the procedure for the isolation of pregnane-3 (α), 20α-diol as previously described. As a result of six chromatographies taken of the above specimens of urine, I presumed that the requisite substance was eluted with 2% methanol in benzene without regard to pregnancy or male urine.
The white coloured and square column or needle shaped crystalline substances were obtained after recrystallization of the crude materials in this fraction in absolute methanol.
These crystalline substances melted at 241.5°C-245.0°C, determined with the microscopic apparatus (Hasegawa).
The absorption spectrums of their H₂SO₄ chromogen exhibited their pearks at 435-440 mμ, and the specimens incubated at 25°C for two hours and twenty four hours, according to the method of Zaffaroni, were almost similar ; the peak at 438 mμ in the visible region and at 300 Imμ/2 in the ultra violet region, which was lower than in the visible region.
With the method used in our department it was investigated further whether these substances yielded acetaldehyde after periodic-acid oxydation or not. The result of this investigation proved that approximately 100% of these crystalline substances yielded acetaldehyde.
As the absorption band exhibited by carbonyl stretching vibration did not appear in the region of the infra red spectrum of these crytalline substances, it was proved that they were not ketonic or that they contained no ketonic substance.
Considering the above investigations, I concluded that these recrystallized substances from 2% methanol in benzene fraction isolated by my chromatographic techique could unquestionably be recognized as almost pure pregnane-3α, 17α, 20α-triol and could possibly be used as standard substances in pregnanetriol studies.

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In our experiments with column chromatography to isolate pregnanediol from the toluene extract of β-glucuronidase hydrolysed urine which had been washed by NaOH and taken from normal eighth month pregnant women, we obtained a crystalline substance in the fraction in which pregnanetriol should have been eluted. Investigation of this crystalline substance, revealed that 61.1% of this substance was acetaldehydogenic steroids (AS).
Together with this result and with the fact that we had obtained pregnanetriol from the urine of women who were given progesterone, we presumed that urinary AS was mutually related with proesterone metabolism.
1. Two normal women were given 100 mg. of progesterone orally every day during the menstrual cycle, and their urinary AS and 17-OH CS were estimated.
AS before administration was found in a slightly greater amount in the progestational phase than in the follicular phase, and was markedly decreased in the ovulatory stadium and just before menstruation. AS markedly increased following administration and a greater amount was found in the cycle after suspension of administration than before administration. The amount of 17-OH CS before administration tended to fluctuate almost parallel with that of AS, decrease following adminstration, and was a little less after suspension than before administration.
2. A normal man and a normal woman were given orally 10 mg. of 17α-ethinyl-5 (10) estraene-19 (β) -0l-3-one (SC-4642 : Searle) every day. The average weekly amounts of AS and 17-OH CS in the male urine before administration were respectively 1.132 mg. a day. In a week of administration his AS markedly decreased and his 17-OH CS tended to decrease a little. In the week following suspension of administration his AS returned to the value administration but the recovery of his 17-OH CS was delayed. As the woman had severe side effects, the administration was ceased after the ninth day. Her AS and 17-OH CS also markedly decreased following administration. After suspension of administration her AS almost recovered but the recovery of her 17-OH CS was delayed.
3. Urinary AS of normal woman was estimated for a period of six days centering around the day when her basal body temperature (B.B.T.) fell. AS was found in the least amount on the day before her B.B.T. fell, but it was markedly raised on the day when her B.B.T. went down.
According to the above investigation we found that AS also existed in the urine of normal pregnant women, that the amount of urinary AS of normal non-pregnant women was mutually related with the ovarian cycle, that progesterone tented to hasten the excretion of AS and restained the excretion of 17-OH CS, and that 17α-ethinyl-5 (10) estraene-19 (β) -o1-3-one restained the excretion of both AS and 17-OH CS.

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The authors decided the optimal conditions for fluorometry of pregnanediol which was intended to be applied for measurement of β-glucuronidase activity.
They measured the fluorescent spectra of pregnanediol reacted with concentrated sulfuric acid, antimonous trichloride and phosphoric acid, respectively.
For fluorometry the dried residue of pregnanediol was treated with 3 ml of concentrated sulfuric acid for 60 minutes in 100°C water bath, then cooled in tap water and allowed to stand for 60 minutes at room temperature. The reaction of preganediol with sulfuric acid proceeded even at room temperature and became stabilized in 45 to 50 minutes. Then the intensity of fluorescence was determined by self-made fluorometer with the exciting light of 436 mμ. Pregnandiol in amounts ranging from 0.01 to 10 μg can be satisfactorily determined.

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Measurements of cerebral hemodynamics and metabolism were made by the nitrous oxide method in 36 diabetic patients,
Considering the factors of blood pressure, the author cmpared the cerebral hemodynamics in diabetics with those in non-diabetics. In the normotensive group, the mean value of cerebral vascular resistance in diabetics was significantly higher than that in non-diabetics. In the hypertenvive group, the mean value of cerebral blood flow in diabetics was significantly lower than that in non-diabetics.
These significnt difference of cerebral hemodynamic changes was observed remarkably in younger group (under 29 years old in normotensive group ; under 39 years old in hypertensive group), and not observed in elderly group.
These facts revealed that cerebral hemodynamic damages in diabetics were observed from younger generation than in non-diabetics.
It is suggested that these cerebral hemodynamic damage is due to the arteriosclerotic change of cerebral blood vessels.
In non-treated group of diabetics, cerebral hemodynamic damage was observed with lapse of duration of diabetes, but not observed in treated group.
The clinical importance of these studies were discussed.
Cerebral glucose metabolism of diabetic patients maintained normal value.

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1) This article summarily discusses the experimental results of the effects of the thyroid hormone on carbohydrate metabolism. The present investigation was carried out by the departmental staffs.
2) Serum P.B.I., I¹³¹ uptake, basal metabolism and cholesterol contents of 62 diabetic patients admitted to our hospital were determined. Two of the 62 patients had hyperthyroidism, of the two one had devloped hyperthyroidism during the course of diabetes mellitus, which was improved by treatment for hyperthyroidism. Generally speaking, severe diabetic patients, having high fasting blood sugar and severe form of glucose tolerance test, were apt to show hypothyroidism.
3) A new method to determine T.S.H. in vitro was recently desingned by appling what I¹³¹ release-inhibiting ratio in calf thyroid slice run paralelled with standard T.S.H. concentration (U.S.P.) (Bakke-Ogura modification for plasma T.S.H. determination). By means of this method the normal plasma T.S.H. level was 0.69±0.51 m J.S.E. per ml.
The patients with moderately advanced and severe diabetes mellitus were apt to show elevated plasma T.S.H., especially in three juvenile diabetic patients.
4) Relatively large number of the patients revealed low serum P.B.I. with elevated T.S.H., as the correlation of these two substances were checked in all the diabetic patients. The investigation is being continued for further clarification.
5) The effects of thryoxine and triiodothyronine on carbohydrate metabolism were investigated in vitro, i.e. these hormones accelerated glucose uptake and glycogen synthesis of rat diaphragm and antagonized insuline effects. However, they are antagonistic in two ways, glycolysis and glycogen synthesis, are accelerated in rat liver slice by the hormones. Thyroxine failed to show any effect on oxygen consumption of brain slice.
6) The thyroid function of the alloxan diabetic rat was investigated subsequently by means of I¹³¹ radioautograph and thyroid function index.* The following results were obtained. The thyroid function was apt to be increased in the early stage, but was generally decreased in the later stage of the alloxan diabetes. With insulin treatment for the lowered thyroid function in the later stage, the function was able to be improved.
* Thyroid function index = I¹³¹ uptake% /thyroid weight / body weight/x 100

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To investigate the participiation of adrenal cortical hormone in the formation of serum proteins, the serum protein fractions in normal rats, in adrenal excised rats, in those given adrenal cortical hormone after their adrenals excised, were studied. The net production of albumin and r-globulin in the liver slices from normal rats, adrenal excised rats, and rats given Cortisone after excision of their adrenals, were also observed by means of quantitative precipitin reaction.

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It is well known that fructose and galactose are easily converted to glucose in the body. Various foods contain fructose and galactose. Therefore, it may be important, from the nutritional standpoint, to evaluate the ability of diabetics to utilize fructose and galactose. Present study dealt with the utilization of fructose and galactose in patients with diabetes mellitus and relation between the utilization of these sugars and the types of disease, age and complication of patients. The results were as follows.
(1) Diabetics showed enhanced interconversion from fructose to glucose and disturbance in the utilization of galactose.
(2) Disturbance of galactose utilization was more marked in patients with severe diabetes mellitus.
(3) The conversion of fructose to glucose was more marked in thin patients and younger patients.
(4) Disturbance of glactose utilization was more marked in older patients and in those with complications.
(5) The elevation of blood pyruvate level following the administration of fructose or galactose was found to be almost the same in diabetics as in normals. However, the rise in blood pyruvate level persisted longer in diabetics than in normal adults.

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