In the previous experiment with 6-dehydro-retroprogesterone and 6-dehydro-proge-sterone, it was concluded that an introduction of 6 : 7 double bond to Δ⁴-3-ketosteroid resulted in the stability of Δ⁴-double bond in the course of the metabolism in vivo. In this experiment, the stability of the Δ⁴-double bond was examined with 6-halogenated Δ⁴-3-ketosteroid, 6-dehydro-6-chloro-17α-acetoxyprogesterone (Δ⁶CAP).
Cold Δ⁶CAP was labeled with tritium by Wilzbach's method. The labeled compound was chromatographed on silicagel and recrystalized 3 times from methanol. Further purification on paper in ligroin-80% methanol system was repeated two times prior the administration to rabbit. The paper chromatographic purification eliminated the contaminated radioactive material which was slightly more polar than Δ⁶CAP.
A rabbit was injected subcutaneously about 90 × 10⁵ cpm (1.97 mg) of 3H-Δ⁶CAP and the other 200 mg of cold CAP daily for 3 days. Seven days' urine was collected, pooled and extracted as in the previous experiment. The extract was chromatographed on 1.2 g Mallinckrodt silicagel, in a 1 cm diameter column.
Radioactivities were eluted stepwise with 100 ml of hexane-chloroform (1 : 1), 200 ml of chloroform and 100 ml each of 2, 5 and 50% methanol in chloroform. Five to six radio-active peaks were eluted from the column and were divided into 3 fractions (A, B and C) as indicated in the Fig. 2. Each fraction was chromatographed on paper, according to the polarity, in (1) ligroin-methanol-water (100 : 80 : 20), (2) benzene-ligroin-methanol-water (70 : 130 : 160 : 40), (3) benzene-ligroin-methanol-water (130 : 70 : 160 : 40) and/ or (4) benzene-methanol-water (100 : 50 : 50). This procedure separated fraction A into 4 (A1, A2, A3 & A4), fraction B into 2 (B1 &B2) and fraction C into 3 (C1, C2 & C3) radio-active compounds. Each compound was purified by rechromatography on paper and the UV absorption spectrum was determined in methanol. Of these metabolites, A1, A2, A3, A4 and B2 had an absorption maximum at 285 mμ indicating the presence of Δ^{4, 6}-3-ketone structure. Other metabolites (B1 C1, C2 and C3) had an absorption maximum at 245 mμ with two shoulders as presented in Fig. 3. Such an absorption spectrum was reported by Dorfman to be characteristic for Δ^{4, 6}-3-ol compound. And this was confirmed, in this experiment, by the fact that a compound obtained by the reduction of Δ⁶-CAP with NaBH₄ in dioxane had almost identical UV absorption spectrum as shown in Fig. 3. It was shown from these results that the Δ^{4, 6}-double bond of Δ⁶CAP was not affected during the course of the metabolism in vivo, but the reduction of C3-ketone without prior reduction of Δ⁴-double bond was demonstrated. Ringold et al reported such an abnormal reduction of C3-ketone in vitro with several Δ⁴-3-ketosteroid having an electronegative substituent at C2, C4 or C6. However, the incubation ofΔ⁶CAP with rabbit liver homogenate, carried out in our laboratory, could not demonstrate the abnormal reduction of the C3-ketone.

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As is well-known, diabetes mellitus is caused by the absolute or the relative shortage of the insulin amount in the body. Considerable changes are expected to exist in the endocrine cells of the pancreas, especially in the ultramicroscopic structure of the beta cells which play an important role in synthesis, preserving and secreting insulin. In fact, in the lightmicroscopic study of the pancreatic tissue of diabetic patients, hydropic degeneration, hyalinization, fibrosis and lymphatic infiltration, etc., are observed. Few electron microscopic observations of the pancreatic beta cells of diabetic patients have been reported, through interesting, due to difficulty in obtaining specimens. Recently, one of the diabetic patients under our long-term observation of the course was complicated by gastric cancer. We were able to obtain fresh pancreatic tissues at the time of operation. We observed them electron microscopically.

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In spite of the fact that weight is the most important factor in calculating the dose of radioiodine for the treatment of hyperthyroidism, a method to calculate the exact weight of the thyroid gland in situ has never been reported.
Since 1952 Allen-Goodwin has introduced a new method of calculating the thyroid weight by using the projecting square of scintigram and a constant value which was obtained from the actual measurement of the thyroid gland.
This method has a serious drawback because the measurement attempts to use the surface obtained by the scintigram in only one plane while the struma exists in three dimensions.
Since 1962 a new approach combining the scintigram with the ultrasonic method can be carried out. First of all, A-scope was used to measure the depth of the thyroid gland which was introduced into Allen-Goodwin formula and the volume of the thyroid gland or struma was calculated as a cylinder or an ellipsoid.
We used this fundamental mechanism to obtain the boundary surface between the struma and neighboring organs and therefore the depth of the thyroid gland or struma was also obtained. The calculation of the volume of the thyroid gland or struma was undertaken to multiply the depth and projection square by the scintigram, in order to calculate an imaginary cylinder or ellipsoid.
Compared with Allen-Goodwin's method, our method is closen to the actual measurement, especially in solid tumors such as adenoma and cancer, which were calculated to almost the same value as the extirpated thyroid gland. As shown in some cases, Allen-Goodwin's method shows a far greater value in calculating diffuse struma such as hyperthy-roidism. Our method of A-scope, in some instances, shows inaccuracy in calculating diffuse changes because the nature of the surface of the gland greatly influences the calculation of the volume.
Because of this difficulty another method was devised in which the thyroid was tomographed by B-scope from the top to the base of the thyroid area in an interval of 3 mm or 5 mm and the square of each section was measured. The volume was calculated correctly by multiplying each square and interval. On the model experiments the hollow plastic structure similar to normal human thyroid was made. Scintigram was carried out on the model filling it with ¹³¹I solution and B-scope tomography was obtained. As a result, considerable accuracy was obtained in measurement of the thyroid gland weight when compared with the actual measurement.
Clinical determination of the thyroid gland weight and the desired doses of radioiodine to treat the hyperthyroidism was then calculated. The administration of radioiodine employed by our new method required smaller doses of ¹³¹I than indicated by the former calculation and was effective for the treatment of hyperthyroidism without causing myxedema.
Two hundred fifty cases of thyroid diseases have been studied by ultrasonic method and thyroidal scintigram, and 30 cases out of them have been examined pathohistologically.
The results of comparative studies among the ultrasono-tomogram, scintigram and histological examination of extirpated thyroid gland are repoted in this paper.
The method of thyroidal scintigram is important to detect the place, shape, size, and abnormality of the thyroid gland. Therefore, this is an excellent procedure to estimate the functional and morphological state of the thyroid gland. However, there is some difficulty in distinguishing between thyroid cancer, chronic thyroiditis and benign goiter. For this purpose it is necessary to determine the characteristics of the tumors by using ultrasonic diagnosis auxiliary.
At present there are some questions on the resolving power of the ultrasonic sound, particulary concerned with attenuation of the ultrasonic sound, In the case of diagnosis of thyroidal diseases by means of the ultrasonic method,

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1) In Ca-free Tyrode solution calcium content of the myometrium dereased. Calcium was thought to be more loosely bound in the progesterone-dominated myometrium than in the estrogen-dominated myometrium.
2) Contractions and action potentials well synchronized in the estrogen-dominated myometrium, but were asynchronous in the progesterone-dominated myometrium.
3) Membrane resting potential of the untreated myometrium on the 21st day of gestation was 39.5 mV. This value was hyperpolarized to 46.5mV in the estrogen-dominated myometrium and to 48.7 mV in the progesterone-dominated myometrium.
4) Estradiol and progesterone in vitro inhibited uterine contractions, but did not alter the resting potential levels.
5) The contractile effect of sparteine sulfate was markedly inhibited in Ca-free Tyrode solution, but to a lesser degree in normal Tyrode solution at low grade temperature.

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During the last few years, many progestational steroids were freshly synthesized and widely used for clinical purposes.
From routine biological assays of these steroids, it appears that slight modifications of the structure resulted in marked changes in their biological activities. Not only progestational activity, but some of these steroids have corticosteroid-like activity and some have androgenic or estrogenic activity.
In this experiment, biological characteristics of progestational compounds with corticosteriod-like activities, and effects of corticosteroids on progestational activities of verious synthetic progestins were investigated.
The progestational activities were determined by the McPhail assay and endometrial Carbonic Anhydrase Activity (CAA) in Clauberg rabbit.
CAA was estimated by the Hirashimizu's modification⁹⁾ of Pincus-Miyake's technique⁸⁾.
As can be seen in Tab. 1, 17α-acetoxy progesterone derivatives, that is 6α-methyl-17α acetoxy-progesterone (MAP), 6-chloro-6-dehydro-17α-acetoxy-progesterone (Δ⁶CAP) and 6-methyl-6-dehydro-17α-acetoxy-progesterone (Δ⁶MAP) were more potent than the other synthetic progestins.
On the other hand, as an indicator of clinical progestational activity, effect of these synthetic progestins on “delay of menses” was tested. The result is shown in Tab. 2, 17α-ethynyL-19-nor-testosterone (ENT) and other estrogenic progestins (i.e. ethynodiol diacetate (EEDDA), norethynodrel (EEO) and Lynestrenol (EEL)) were more effective than 17α-acetoxy-progesterone derivatives. This result markedly differed from that of the above-mentioned animal assays. It is considered that the inherent estrogenic activity in 19-nor-testosterone derivatives impedes the progestational response in Clauberg rabbit, but acts with advantage on delay of menses. 17α-acetoxy-progesterone derivatives have a cortisone-like activity. Some investigators reported that in rats, administration of MAP resulted marked adrenal atrophy and thymus involution. Large doses administration of Δ⁶ MAP and Δ⁶ CAP also caused thymus involution.
Therefore, the possibility of potentiating effect with cortisone-like activity on the progestational response in Clauberg rabbit was investigated in the following experiment.
While no endometrial response was found with corticosteroid alone, the administration of corticosteroid in combination with progesterone resulted in a more marked endometrial response in the Clauberg rabbit than that with progesterone alone. The more the corticosteroid was combined, the higher the endometrial response was (Fig. 1). The potentiating effect of prednisolone was significantly higher than that of hydorcortisone acetate. And the potentiating effect of added corticosteroid was observed more markedly on endometrial CAA than on endometrial proliferation in Clauberg rabbit.
As compaired with the assay using progesterone alone, only one thirtieth of progesterone was required in CAA. assay where 25 mg of hydrocortisone acetate was simultaneously administered. These effects of corticosteroids were also observed with some synthetic progestins, while only a very slight effect could be obtained with 17α-ethynyl-4-estrene-38, 17β-diol-diacetate (Table 5).
To potentiate the effect of progesterone, however, simultaneous administration of corticosteroid was not the only method, since corticosteroid administration in the course of estrogen priming or the single injection in progesterone treatment resulted in similar effects as shown in Fig. 2. CAA in the adrenal, kidney, liver and blood were neither influenced by progesterone administration nor by the corticosteroid combination.
Incubation of liver and endometrial tissue with or without cortiscoteroid resulted in no rise in CAA in these tissues.

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Estrogens are frequently used for clinical purposes. Estradiol (ED-17β), which is a natural estrogen, however, is inactive when administered orally.
The activation of orally administered ED-17β has been supposed to take place in the liver. Ethynyl estradio1-17α (EED-17α) which is a synthetic estrogen is effective in both oral and subcutaneous administration.
This study was undertaken to investigate the metabolic differences between ED-17β and EED-17α in rabbit.
³H-EED-17α was orally or subcutaneously administered in a large or a small dose. ¹⁴C-ED-17β was administered in a large or a small dose.
After the administration, urine samples were pooled every 24 hours and estimated by counting in the liquid scintillation spectrometer. In the small doses, administration of ED-17β, about 97% of radioactivity was found to be excreted in the urine during a 2-day period. When administered in a large amount, however, excreted radioactivity was only 20.1%. In the ³H-EED-17α administration, urinary excretion of radioactivity tended to be delayed compared with ¹⁴C-ED-17β. No remarkable difference in the excretion of radioactivity was observed regardless of mode of administration. The urine sample was extracted with chloroform to give free fraction of excreted radioactivity. The urine was then incubated with β-glucuronidase for 36 hours and extracted with chloroform (glucuronide fraction). Residual urine was extracted with ethyl acetate at pH 1 after saturation with ammonium sulfate. The organic extract was incubated for 36 hours. After incubation, organic extract was washed with 8% NaHCO³ water. (solvolysis fraction)
Finally, washings were hydrolyzed with HCl in the boiling water bath and extracted with ethyl acetate (hot acid hydrolysis fraction). With ³H-EED-17α, most of the radioactivity appeared in the sulfate fraction. ¹⁴C-ED-17β administration resulted in high levels of radioactivity in the free and glucuronide fraction.
The extract of each fraction was subjected to partition chromatography on alumina column. Elution was carried out by the stepwise addition of increasing concentration of methanol in benzene. Three peaks of radioactivity, A, B and C were shown in the glu-curonide fraction of ¹⁴C-ED-17β.
Radioactive peak B, which was highly eluted in 0.5% methanolbenzene, was similar to ED-17α on paper chromatography, thin layer chromatography (TLC) and gas liquid chromatography (GLC). Constant specific activity was obtained on recrysatllization with authentic ED-17α.
In solvolysis fraction, however, peak B disappered from the distribution pattern and radioactivity was present mainly in 50% methnol-water fraction. Because of its ethyl acetate soluble nature, this radioactive material is most likely 17α-ED-17β-N-acetyl glucosamine conjugate.
Peak A which eluted in 0.3% M.B. was identical with that of authentic estrone. In large-dose administration, the ratio of radioactivity incorporated into estrone fraction was higher than in small-dose administration.
Peak C corresponded to estriol in the column chromatographic behavior was eluted with 5% methanol in benzene. This material which had identical Rf. value with authentic estriol on paper chromatography was eluted and subjected to TLC. Radioactive spot was not identical with estriol, but with 16, 17-epi-estriol. However, the absolute amount was too small for further study.
On large-dose administration of ³H-EED-17α, the radioactivity was eluted mainly with 0.3% methanol in benzene as EED-17α.
The sulfuric acid chromogen spectra and ultra violet absorption spectra of this peak were identical with those of authentic EED-17α. Following addition of unlabelled EED17α to the isolated radioactive fraction,

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A new simple method for the determination of grouped neutral steroid glucuronide by gas-liquid chromatography was described. The new method was based upon the utilization of 1% QF-1 column for the steroid residues which have been treated by NaBH₄ and NaI0₄ following Norymberski's method. The method was carried out as follows :
1. 10 ml of urine was taken from pooled 24-hour urine.
2. Reduction by NaBH₄
3. Oxydation by NaIO₄
4. Extraction with ethyl ether.
5. Washing with water.
6. Dry down and evaporation.
7. Gas-liquid chromatography.
The gas-chromatograph was type GC-1B with ion flame detector produced by Shimazu Seisakusho Co. in Japan. With this procedure, the neutral steroid glucuronide was separated into four groups.
I. 11-deoxy-C₁₉ steroids
II. 11-deoxy-C₂₁ steroids
III. 1 1 -oxy-C₁₉ steroids
IV. 11-oxy-C₂₁ steroids
These four groups were separated according to the specific affinity of QF-1 column to steroid keton or hyroxy and especially to 11-oxy steroid. The identification of separation was examined with sixteen authorized pure steroids and the products produced by reduction and oxydation.
When the steroid residue described above was acetylated with acetate anhydrous and pyridine before injection to gas-chromatograph, the acetylated neutral steroids were separated into three groups
I. 11-deoxy-neutral steroids
II. Pregnandiol
III. 11-oxy-neutral steroids
These three groups were separated through QF-1 column, since with QF-1 column the acetylated hydroxy in C-17 position showed similar retention time with ketone in C-17 position and then 17-KS and 17-KGS made two groups which were 11-oxy, and 11-deoxy, pregnandiol made another group.
By this method, one may estimate easily eleven oxygenation rate in a clinical course : the procedure of the determination is simple and can be finished in 5 or 6 hours. The reproducibility is sufficient and the results can be kept as a chart.

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With the gaschromatographic technique which is described in Chapter 1, four grouped neutral steroid glucuronide and three grouped neutral steroid glucuronide were determined in each twenty cases of normal female follicular phase and luteal phase, normal male and castrated female respectively, and in sixteen cases with endocrinopathy. As the dynamic test, the responses to ACTH-Z, SU-4885 and dexamethasone were examined in two cases with endocrinopathy.
As a result, the mean values and standard deviations of four groups were as follows :
No. of cases I II III IV
Normal female20Mean 26.7 14.7 6.9 51.7%
prol. phase S.D. 8.6 5.1 5.9 11.1
Normal female 20 Mean 18.8 33.6 10.6 37.0
secr. phase S.D. 6.0 9.2 6.5 9.4
Normal male 20 Mean 30.0 18.1 6.1 45.7
S.D. 10.8 7.6 6.4 10.8
Castrated female 20 Mean 29.1 12.0 11.1 47.9
S.D. 17.9 10.0 6.7 18.8
(I 11-deoxy-C₁₉ steroid, II=11-deoxy-C₂₁ steroid, III=-11-oxy-C₁₉ steroid, IV = 11-oxyC₂₁ steroid). Normal male and normal female follicular phase showed the same pattern on scattergram in four fractions. In these two, 11-oxy-C₂₁ steroid was most eminent and 11-deoxy-C₁₉ steroid, 11-deoxy-C₂₁steroid and 11-oxy-C₁₉ steroid followed it, in this order. In the cases of normal female luteal phase, the order was reversed in 11-deoxy-C₂₁ steroid and 11-deoxy-C₁₉ steroid, and this resulted from increased excreation of pregnandiol (5βpregnan-3a, 20a-diol). In the cases of castrated females, the pattern of four fraction was the same as the males and normal female follicular phase, but it showed a broader dispersion than these two.
The mean values and standard deviations of three grouped method were as follows :
No. of cases I II III
Normal female20Mean 39.4 10.1 50.4%
prol. phase S.D. 7.6 4.1 9.5
Normal female 20 Mean 36.4 26.0 37.6
secr. phase S.D. 10.3 10.6 11.8
Normal male 20 Mean 46.2 5.1 48.7
S.D. 8.3 2.5 8.7
Castrated female 20 Mean 36.6 12.0 51.3
S.D. 21.5 7.2 24.6
(I = 11-deoxy neutral steroid, II = Pregnandiol, III =11-oxy-neutral steroids) The second fraction (pregnandiol) was remarkably increased in luteal phase and in pregnancy. This three-grouped method would be usable as a simple method of determination of pregnandiol. Eleven oxygenation rate was calcurated dividing fraction III by fraction I, and the mean values and standard deviations were as follows :
Normal femal Normal female Normal male Castrated female prol. phase secr. phase
No. of cases 20 20 20 20
Mean 1.35 1.17 1.12 2.40
S.D. 0.49 0.63 0.40 1.97
In the cases with endocrinopathy, a high eleven oxygenation rate was shown in the cases with Simmonds' disease, Cushing's syndrome and male eunuchoidism, and a relatively low rate was shown in the cases with hypoadrnocortism. After administration of SU-4885, 11deoxy-C₂₁ steroid in four-grouped method and 11-deoxy-neutral steroids in three-grouped method increased remarkably. After administration of ACTH-Z, 11-oxy-C₂₁ steroids in fourgrouped method and 11-oxy-neutral steroid in three-grouped method increased contrary. By suppressing adrenals with dexamethasone, relative decreasement of 11-oxy-G₂₁ steroid and 11-oxy-neutral steroid was shown.
It was concluded that this method was rapid and simple for determinating neutral steroid glucuronid : one is able to easily determine the ratio of C₁₉ steroid and C₂₁ steroid, and 11-oxygenation rate of neutral steroid glucuronide in clinical course.

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