In the previous report, the conversion of 17α-ethynyl-4-estrene-17β-ol to 17a-ethynyl-19-nortestosterone was demonstrated in rabbit liver homogenate. In this experiment, conversions of 17α-ethyl-4-estrene-17α-ol and 17α-allyl-4-estrene-17α-ol to the corresponding 3-keto compounds in vitro were examined with rabbit liver slices.
Liver obtained from a female adult rabbit was cut into slices 1 mm thick with a thin blade. Each vessel contained 2 mg of steroid in 0.5 ml of propylenglycol, 50 ml of Krebs Ringer Phosphate (PH 7.2) and 5 g of liver slices. After 4 hours incubation under air, the incubation medium was extracted 2 times with equal volume of methylene chloride. It was then washed with a small amount of water and evaporated in vacuo. The extract was transferred with benzene to 1 cm column containing 6 g of Brockmann alumina. After preliminary wahsing with 100 ml benzene, polar metabolites were eluted with 1.0% methanol in benzene. This was evaporated to dryness and chromatographed on paper in benzene-ligroin-methanol-water (70 : 130 : 160 : 40). Metabolites with a, β-unsaturated oxo groups were detected with UV light and were eluted with methanol. After rechromatography on papeer, the UV absorption spectrum of each metabolite was determined. Gas-chromatography was carried out at 220°C on the Shimazu model GC-1B with 150 cm columns containing 1.5% SE-30, chromasorb W. In the experiment with 17α-ethyl-4-estrene-17β-ol, two metabolites (M1 and M2) were detected on paper. M1 was identified as 17α-ethyl-19-nortestosterone from the UV absorption maximum at 244 miμ, RI values on paper and RT on gas chromatography. M2 was suggested to have Δ⁴-3-keto structure from the UV absorption maximum at 241 mμ but the complete structure was not yet known. Incubation of 17α-allyl-4-estrene-17β-ol gave a metabolite which had UV absortpion maximum at 244 mμ, e, indicating the presence of Δ⁴-3-keto structure. While the authentic sample of 17α-ally1-19-nortestosterone was not on hand, it was highly probable that the metabolite was this compound from the retention factor on gas chromatography. All these data indicated the conversion of 3-deoxosteroid to the corresponding 3-keto-compound in vitro. The in vivo conversion of 3-deoxosteroid was demonstrated in our laboratory with 17α-ethynyl-4-etrene-17β-ol in rabbits and in humans.

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Various factors were evaluated to measure the serum prealbumin by a single radial immunodiffusion. The serum prealbumin and the maximum binding capacity of thyroxine-binding prealbumin (TBPA) in normal males and females were also determined.
Rabbits were immunized 2 times with a homogenate of complete adjuvant-human prealbumin (containing 0.3 mg) mixture, a booster dose of 0.3 mg of human prealbumin was intravenously administered after 14 days, and on the fifth day after the terminal immunization, a blood sample was collected from A. Carotis. An antibody titer showed approximately 1 : 20,000 by the method of Boyden.
In the method of radial diffusion here studied, the antibody was incorporated in various concentrations in a 1.5% agar made in 0.1 N Michaelis buffer of PH 8.6 containing 0.1 % sodium azide.
The thickness of the gel was uniformly set at 1.5 mm by pouring 8 ml of the warm fluid agar-antiserum mixture into an empty immunoplate (Behringwerke). After solidification of the gel, wells of 2.1 mm in diameter were punched out, and filled with 2μl of antigen solutions by a microsyringe.
Results are summarized as follows :
1) It was adequate that antiserum was diluted in 5-7%, human serum undiluted or diluted in 1 : 2 with a saline. The sensibility, the visible precipitate extended down to as little as 0.008 μg, eg or 4μg ml of antigen in 2% dilution of antiserum.
2) There was a significant linear relationship between the serum prealbumin concentration and the maximum binding capacity of TBPA.
3) In adults, the serum prealbumin was significantly higher in the male than in the female, the aged persons had no sexual difference in the serum prealbumin and had the lower prealbumin concentration than adults.
It was suggested that the serum prealbumin concentratoin could be influenced by gonadal steroids. 4) The possibility that approximately 8 μg of thyroxine could be bound per mg of the serum prealbumin was suggested, the number of binding sites per molecule was calculated to be 0.58.

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The present paper concerns with the determination of plasma free 11-OHCS in normal healthy persons and aged patients suffering from cerebrovascular diseases. The diurnal and daily variations in plasma free 11-OHCS levels were studied. Experiments were performed on 47 healthy laboratory workers and medical students aged from 18 to 26 years and on 65 patients with cerebrovascular diseases aged from 36 to 87 years.
Plasma free 11-OHCS was estimated by the modified method of De Moor's fluorometric method which was reported previously. (Igari. J., et al. Jap. J. Clin. Path. 15 (8) : 591-594, 1967)
The results were as follows :
1) The mean levels of plasma free 11-OHCS which collected from 47 normal persons were 16.4±4.7 pg./100 CC. and the range was from 5.5 to 20.0μg./100 CC. It has been reported that the normal values ranged from 9.5±2.6 pg./100 CC to 21.9±4.7 μg./100 CC by several workers.
The results of 65 cases, all aged patients, showed 13.5±5.0 μg./100 CC. It was lower than that of normal persons. (P<0.05). According to the results, it was suggested that the plasma free 11-OHCS level might vary by aging in aged patients.
2) In 11 healthy laboratory workers, the plasma free 11-OHCS levels at 9 AM were determined duriing 10 days. The significant daily variation was noted in all cases, but no fixed pattern was observed in daily individual variations of the plasma free 11-OHCS levels.
3) For the determination of the diurnal variations in plasma corticosteroids, plasma free 11-OHCS levels were estimated on samples collected at 9 AM and 5 PM in 8 aged patients. The levels were higher at 9 AM than at 5 PM in 6 out of 8 patients, but the levels at 5 PM showed moderate elevation in 2 of these patients.
A more detailed study of the diurnal variation was carried out by taking samples at 6-hour intervals over a 24-hour period in 3 aged patients. The maximal level was obtained at 6 AM and the minimum level was obtained at midnight in all 3 patients. It was well established that the plasma corticosteroids showed a maximum level between 5 AM and 9 AM and showed a minimum level at midnight. The results were similar to that of other authors.
These findings indicate that clinical significance of the levels of plasma free 11-OHCS should be evaluated by taking these diurnal and daily variations into consideration.

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In this report, using normal female Wistar rats, ovarian L-LAP and L-CAP activities, L-LAP isozyme patterns and the influences of gonadotropin on them were investigated to clarify the effects of the hormone upon the protein metabolism in the ovary. Their ovaries were removed immediately after the rats were killed by cutting a carotid artery to homogenize them in a glass-homogenizer.
Torigoe-Wada's method using the reaction between p-diaminobenzaldehyde and β-naphthylamine was applied to estimate the aminopeptidase activities. And in order to separate the L-LAP isozymes of ovarian homogenate, starch block electrophoresis (200 V., 4 mA., 15 hours) with veronal veronalsoda buffer solution (pH : 8.6, /μ=0.05) was utilized.
The L-LAP and L-CAP activities were showed as them per one rat, and their activities of I unit had meant that under the standard condition the enzymic activities hydrolysing their substrates to liberate 1 mg. of β-naphthylamine.
The results obtained are summarised as follows.
(1) The L-LAP could be separated into 3 isozymes by the electrophoresis, the one ran slowly to anode (I called the L-LAP Isozyme I), the second ran fast to anode (the Isozyme II) and the third ran to cathod (the Isozyme III).
(2) As normal rats grew up, both the L-LAP and L-CAP activities were evidently increased without showing any parallel fluctuation of the two enzymes, and the L-LAP isozyme patterns changed slightly, and the L-LAP Isozyme III was revealed markedly only in mature ovary.
(3) After the sexual cycle was initiated, both the L-LAP and L-CAP activities fluctuated in a certain pattern responding to the ovarian cycle, that is, they reached maximum at metestrus, then markedly decreased at diestrus, moderately increased again at proestrus and decreased again to reach minimum at estrus. And in these groups the fluctuations of two enzymes were not paralles either.
The L-LAP isozyme patterns were altered moderately responding to the ovarian cycle, that is, the Isozyme I and the Isozyme II were usually present in all groups, but the Isozyme III was remarkable only on metestrus (at the phase of corpus luteum formation).
(4) The single subcutaneous injections were carried out to each group consisting of 5 or 3 rats aged 3 weeks (40-50 gm. weights). The dosages and kinds of gonadotropin were PMS 3 I.U., HCG 5 I.U. or both of them (small doses), PMS 30 I.U., HCG 50 I.U. or both of them (massive doses). And 48 or 96 hours after the administration their ovaries were removed to examine their aminopeptidases.
Both L-LAP and L-CAP activities were increased by the administration of gonadotropin, and their increasing ratios were larger after the administration of massive doses than the small doses in all groups. Their activities 96 hours after the injection were greater than those of 48 hours after the injection in all groups. Their activities in rats treated with PMS were greater than those in rats treated with HCG at both stages of observation. The L-LAP isozyme patterns changed considerablly responding to the doses or kinds of gonadotropin injected. And the L-LAP Isozyme III was detected only in the groups treated with massive doses of HCG especially with massive doses of HCG and PMS simultaneously.
The data mentioned above would appear to support my suspicion that if I selected a suitable dose of gonadotropin, proper kinds and a combination of them, and an adequate method for the administration, it would be possible to make the characters of the ovarian aminopeptidase in immature rats approach those in mature rats by the administration of gonadotropin.
(5) The hypophysectomies of the 4-week-old female rats were carried out by Koyama's method under nembutal anesthesia. On the 3rd day after operation, the massive doses of gonadotropin were applied in the same way to examine the effects of gonadotropin on the ovarian aminopeptidase to compare them with those of normal immature rats.

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It has been indicated that 17α-ethynyl-4-estrene-17β-ol shows biological effects very similar to 17α-ethynyl-19-nortestosterone. The conversion of the former to the latter was reported in vitro with rabbit liver homogenate. In this experiment, the conversion was studied in vivo with rabbit.
Urine collection was made for 4 days after injection of ³H-17α-ethynyl-4-estrene-17β-ol which was prepared from the cold compound by Wilzbach's method. About 15% of the administered radioactivity was excreted in the first day urine and about 35% during 5 days. Almost all the radioactivities were present in the urine in the conjugated form and was extracted by glucuronidase hydrolysis or solvolysis. The extracted materials were separated on alumina column. The stepwise elution of the radioactivity was carried out successively with benzene, 0.1% methanol in benzene, 0.3% methanol in benzene, 0.5% methanol in benzene, 1.0% methanol in benzooe, 5% methanol in bezene and finally with 30% methanol in benzene. Four radioactive peaks named A, B, C and D were eluted from the column with benzene, 0.1% methanol in benzene, 0.3% methanol in benzene and 5% methanol in benzene respectively. The radioactive peak B was eluted in similar fashion as 17α-ethynyl-19-nor-5a-androstane-17β-o1-3-one from the column and the Rf value on TLC in benzene acetone (4 : 1) system was also identical with the reference compound. Rf value on paper chromatography, color reaction on paper, retention time in G.L.C., sulfuric acid chromogen spectrum and several chemical reactivities also indicated the identity of this compound with 17α-ethynyl-19-nor-5a-androstane-17β-ol-3-one. Peak C was identical in the same criteria with 17α-ethynyl-58-19-norandrostane-3a, The compound was easily acetylated with acetic anhydride-pyridine at room temperature, indicating the presence of primary or secondary hydroxy group. The oxidized product of peak C with aqueous chromic acid was identified as 17α-ethynyl-5β-19-norandrostane-17β-ol-3-one from the chromatographic mobilities and several color reactions. Chemical structures of radioactive peak A and D were not well determined. However, several re-activities of peak D were very similar to that obtained from the rabbit injected with 17α-ethynyl-19-nortestosterone indicating that the radioactive peak D was a metabolite of the latter compound. All these data indicated that 17α-ethynyl-4-estrene-17β-ol was first metabolized in vivo to 17α-ethynyl-19-nortestosterone and then underwent further metabolism. In the next section, the metabolite in the blood after intravenous administration of 17α-ethynyl-4-estrene-17β-ol was examined. The blood was collected from the femoral artery for about 60 minutes following injection. Free steroid was extracted with chloroform. The extract was chromatographed on paper in benzene-ligroin-methanolwater system. Three radioactive peaks were observed on paper. The first radioactive peak was identified as 17α-ethynyl-4-estrene-17β-ol from the chromatographic mobilities and color reactions. The second radioactive peak was identified as 17α-ethynyl-19-nortestosterone from the chromatographic mobilities. The third radioactive peak was identified as an identical metabolite with radioactive peak D in the experiment with urinary metabolites. Experiment with the metabolite in blood clearly indicated the conversion of 17α-ethynyl-4-estrene-17β-ol to 17α-etilynyl-19-nortestosterone in vivo.

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