In our previous report it was suggested that urinary hCG is composed of two components. One is dominant in LH-like activity and the other is dominant in FSH-like activity, and the ratio of these components differs according to the stage of pregnancy.
In order to clarify the nature of plancental hCG, this study has been performed.
The crude hCG fractions extracted from trophoblastic tissue of normal placenta and hydatidiform mole by the percolation method of Bates were purified by the combination of Sephadex gel filtration, CM-C and DEAE-C chromatography. Two purified hCG_s (hCG-A and hCG-B) were obtained.
Biological activities of components were measured by an ovarian weight method in immature female rats (λ = 0.176), using the Second International Standard hCG as a reference standard preparation.
Examined by polyacrylamide disc gel electrophoresis, hCG-A and hCG-B showed single bands.
Specific biological activities of hCG-A and hCG-B from normal tissue were both 8,200 IU, but those of hydatidiform mole were very high, namely, hCG-A had an activity of 23,000 IU and hCG-B 8,600 IU.
Moreover, the hCG_s isolated from molar tissue contained much higher hexose, but much less hexosamine, than hCG_s from normal trophoblastic tissue. hCG-A, which contained more hexose, had less sialic acid than hCG-B.
In Ouchterlony immunodiffusion, hCG-A and hCG-B made precipitation lines respectively with anti-hCG-A serum and anti-hCG-B serum, and in immunoelectrophoresis in agar hCG-A and hCG-B formed single precipitation lines with corresponding antiserum.
In addition, hCG-A was neutralized biologically only by anti-hCG-A serum, and the same was observed concerning the hCG-B and its and serum system, using an ovarian weight method above mentioned.
Then 200 IU of hCG-A or hCG-B was injected into the hypophysectomized immature female rats and the biological properties of two hCG_s were examined.
As a result, hCG-A promoted the normalization and hypertrophy of the interstitial cells and did not stimulate any follicular development, but hCG-B stimulated the follicular growth only.
At the same time, although hCG-A had a remarkable action to induce the ovarian Δ⁵-3β-ol dehydrogenase activity in the follicular walls and in the interstitial cell mass, hCG-B could not activate the enzyme activity.
In the other experiment, after the injection of hCG-B into the hypophysectomized immature rats, the marked increase of ³H-thymidine uptake of granulosa cells of the growing follicles was recognized, but with hCG-A the uptake was not so clear.
On the other hand, hCG-A promoted the ovarian phosphorylase activity and elevated the serum progesterone level of the hypophysectomized rats.
From the above experimental results, it was concluded that placental hCG is composed of two components : hCG-A (human chorionic luteinizing hormone : hCLH) with stimulating effect of interstitial cell normalization and its hypertrophy and with promoting effect of ovarian steroidogenesis, and hCG-B (human chorionic follicle stimulating hormone : hCFSH) with follicle stimulating effect only. They are different from each other not only biologically but also immunologically and chemically.
In an attempt to determine the mechanism of production and secretion of these two hCG_s in the chorionic tissue and to investigate their hormonogenesis in placenta, we decided to cultivate the chorionic tissue in vitro and to make the biosynthetic labeled hCG_s. As the first step of this work, the possibility of organ culture of chorionic tissue was examined in the synthetic medium “199” without serum.
The results of this study are as follow :
1) During cultivations of the chorionic tissue, hCG concentration in the media increased remarkably, estimated not only by bioassay with the ovarian weight method in immature rats,

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III Defect of Insulin Action on the Hepatic Enzymes in Obese Hyperglycemic Mice.
Previous findings in our laboratory suggest that insulin exerts its effect on the hepatic metabolism through the rapid alteration of some enzyme activities and afford some evidences to believe that the primary site of insulin action would be a “channeling” of metabolic flux, presumably during glucokinase or pyruvate kinase reaction. If the mechanism of “channeling” is impaired in some way, the direct effect of insulin on the hepatic metabolism should be abolished and this would result in the decrease of glucose tolerance in spite of the presence of insulin in excess. Thus, diabetes with hyperinsulinemia might develop.
To clarify this hypothesis, the direct effect of insulin on the hepatic metabolism in genetically obese hyperglycemic mice was studied, since these animals show several clinical features resembling to those seen in human obese adult-onset diabetics with hyperinsulinemia.
Obese hyperglycemic Bar-Harbor mice (C57BL/6J-ob) originally derived from the Jackson Laboratory at age of 4-5 months old and also 7-9 months old were used. Total evisceration except the liver was performed by the technique previously reported. Glucose (550 mg/kg/hr.) or glucose with insulin (1.5 or 3.0 u/kg/hr.) was infused through portal catheterisation for 20 minutes and the activities of key glycolytic and gluconeogenic enzymes were measured. The results obtained were as follows :
1. The activities of key glycolytic and gluconeogenic enzymes in the young obese mice were several fold higher than those of lean litter mates.
2. In the lean mice, the activities of key glycolytic enzymes were significantly elevated by the administration of insulin, whereas the activities of key gluconeogenic enzymes were not influenced by the hormone.
3. In the obese mice no significant effects of insulin on the enzyme activities were observed.
4. From analysis of the ratio of key glycolytic enzyme activity to the corresponding key gluconeogenic enzyme activity, it might be concluded that insulin exerts its direct effect on a channeling of metabolic flux between glucose and glucose-6-phosphate.
From these results, a possible mechanism of development of diabetes with hyperinsulinemia was discussed in some detail. (Original is written in Japanese.)

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The chemical structure of TRH (Thyrotropin Releasing Hormone) from the purified extract of pig hypothalmus has been recently determined, and since then synthetic TRH has been prepared in several laboratories. It has been reported that synthetic TRH stimulates the release of TSH from the pituitary of the mammalian species. However, it is obscure whether synthetic TRH has a stimulating effect on nonmammalian species. Therefore, in this paper the effect of synthetic TRH on the metamorphic changes of tadpole is reported.
(Bullfrog) Tadpoles were collected from the same place. When tadpoles grew up to ca 3.5 cm in whole length, they were used for the experiment. During the course of the experiment, no diet was given. No decrease in tail length and almost no appearance of fore and hind limbs was observed in the control group injected with saline under these conditions for 10 days.
When 200 μg of synthetic TRH (acetate form of L- (Pyro) Glu-L-His-L-Pro (NH₂)) was injected intraperitoneally 4 times every other day, the decrease in tail length and the appearance of fore and hind limbs were observed to be as significant as the groups given thyroxine or TSH.
When synthetic TRH dissolved in water (1 ng/ml, a final concentration) was given orally, these metamorphic changes were not observed.
From these results, it is supposed that synthetic TRH is effective on amphibian metamorphosis by the direct stimulation of the amphibian pituitary.

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The pathogenesis of essential hypertension with a supressed plasma renin activity (PRA) remains obscure.
In the present study, the pattern of electrolyte homeostasis during four-days' salt restriction, and the effects of spironolactone on the blood pressure and PRA in patients with essential hypertension with a suppressed PRA were compared with that obtained by identical studies performed on the patients with proven primary aldosteronism and on the normal controls. Fifty-three patients were studied, including 38 patients with benign essential hypertension, 5 patients with primary aldosteronism, and 10 patients with normotension.
After a furosemide test, PRA was determined while the patients were in the recumbent position on a diet containing 200 mEq of sodium a day and in the upright posture after they had been on a diet containing 25 mEq of sodium a day for 4 days. The spironolactone was given to the patients with a suppressed PRA in a dose of 200 mg a day and to the patients with primary aldosteronism in a dose of 300 to 400 mg a day. The results were as follows.
1) Among 38 patients with benign essential hypertension, 13 patients (34 percent) failed to respond at all with an increased PRA to the furosemide administration, while the remaining 25 patients had normal PRA response.
2) Of 13 hypertensive patients showing a suppressed PRA response to the furosemide administration, 12 patients failed to respond with a significant increase of PRA also to the salt restriction, and only one patient showed a normal PRA response to the salt restriction.
3) In five of 12 patients with a suppressed PRA to both tests (Group A), the pattern of sodium excretion following salt restriction resembled that of the patients with primary aldosteronism; that is, there was an abrupt decrease of sodium excretion with a significant decrease of potassium excretion. The remaining seven patients (Group B) showed the same sodium excretion pattern after salt restriction as that of normotensive patients.
4) All the patients of group A became normotensive within 2 weeks after spironolectone therapy with significant increases in the serum levels of potassium and PRA; the patients of group B had an insignificant reduction of blood pressure without any changes in the serum levels of potassium and PRA for 7 sweek after spironolactone therapy.
5) All patients with a suppressed PRA showed normal levels of aldosterone in urine.
From these results, it was suggested that there were at least two types of hypertension with a suppressed PRA, one was mineralcorticoid (other than aldosterone) dependent (Group A) and the other one was not (Group B).

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Fundamental and clinical studies on the newly developed thyroid function test “Reso-Mat ETR” were performed.
The test procedures were rather simple and needed one more manipulation, micropipetting of serum, than those of Res-O-Mat T₄ test, and the test could be interpreted as the determination of serum T₄ concentration in test serum in the presence of test serum itself.
The test results were expressed as 'Effective Thyroxine Ratio', a fraction of serum T₄ concentration similarly determined in normal serum. Therefore the effects of incubation temperature and incubation time were not crucial but incubation at room temperature for not less than 1 hour seemed optimal. Variability in a kit using pooled serum was only 0.8% and reproducibility among kits was also satisfactory. To clarify the principle of the test, the effects of various serum volumes were studied in various thyroid status. Irrespective of the amounts of added serum or thyroid status, there found almost linear correlation between the radioactivities retained in the vial (bound T₄) and calculated ratio of total T₄ over total TBG in the system. Total T₄ were the sums of T₄, standard T₄, ethanol extracted T₄ from test serum and additional T₄ derived from micropipetting of test serum, while total TBG included both TBG in standard serum and micropipetted test serum. Some of these values were obtained from experimental results (T₃-RU and T₄ concentration) and the others were calculated theoretically.
From the above observations, the test was found not to be the direct measurements of free T₄ but those of T₄/TBG ratio. However, observations of ERT values in normal pregnant women under various amounts of added serum revealed that obtained higher values without additional serum could be normalized by addition of small amounts of serum (0.0025-0.005 ml), and further addition resulted in lower values. These finding could be ascribed to the excessive free TBG in additional serum of pregnant women.
In summary for the above fundamental observations, the test was concluded to be a unique, simple and reliable method of indicating thyroid status irrespective of the TBG conditions.
Res-O-Mat ERT tests were appraised for 239 clinical cases with various conditions. The test results were as follows : euthyroid 82 cases 0.987±0.067 (S.D.) normal range 0.85-1.12 (mean ±2 S.D.), hyperthyroid 26 cases 1.371±0.132, pregnant Graves' under MMI 2 cases 1.15 and 0.94, hypothyroid 17 cases 0.826±0.048, normal pregnant 8 cases 0.950±0.041. Good differentiation of hyper- or hypothyroid cases from euthyroid cases were obtained and pregnant cases also showed reasonable values.
In 104 cases with Graves' disease after radioiodide treatment (up to 15 years), deter-mined ERT values matched well to their clinical status and chronological observations clearly demonstrated the delayed effect of radioiodide as gradual and consistent decrease in ERT values.
In the 70 cases T₃RU and serum T₄ concentration by competitive protein binding analysis were also determined, and free T₄ Index (FTI) was calculated according to our RU previously reported formula T₃RU×T₄/I-0.6T₃RU.This FTI had been proved to correlate straight-linearly with measured free T₄ in serum.
Among these 3 in vitro thyroid tests, ERT were found best in the correlation with calculated FTI (r=0.916), though their correlation was not straight linear but curvy-linear. ERT also showed higher correlation with both T₃RU (r=0.861) and Ti (r= 0.896) than the correlation between T₃RU and T₄ (r=0.913).
In conclusion,

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In the previous reports, it was demonstrated that hPL had the remarkable promoting action on lipolysis, glucose uptake of the adipose tissue and insulin secretion in vivo.
The purpose of this paper is to describe the results of the studies on the biological action of this hormone during pregnancy in detail.
First, in the preliminary experiment, the author examined the effect of hPL on the foetal growth in utero.
Namely, by the continuous administration of the various doses of hPL to the pregnant rats, the significant increase in the content of glucose, free fatty acid (FFA), triglyceride (TG), total nitrogen (T-N) and body weight of the foetuses was noted.
At the next step of experiment, in order to know this effect of hPL in the foeto-placental maternal system, the maternal adipose tissue was labeled with oleic acid-l-C₁₄, and four hours after the administration of hPL, C₁₄-FFA and cold FFA of maternal blood and foetal carcass, and C₁₄-TG of maternal fat tissue and foetal carcass were measured.
C₁₄-FFA and cold FFA of blood and C14-TG of fat tissue increased remarkably in the mother rats treated with hPL and, at the same time, the foetuses from the hPL treated mother rats showed also the marked increase in C₁₄-FFA and C₁₄-TG content.
In the other experiment, the pregnant rats on L₁₆ (16th day of pregnancy) were injected with hPL following the pretreatment with D-glucose-C₁₄, and then C₁₄-glucose and cold glucose in the foetal liver were estimated.
As the result, the concentration of C₁₄-glucose and cold glucose of the foetal liver was about two times as high as that of the foetuses from the control rats.
On the other had, the suspected immediate action of hPL on the foetuses was studied also with direct injection of it to the foetuses in one uterine horn using them in another horn as the control.
Furthermore, the author measured and compared the activities of lipoprotein lipase (L.P.L.) and hormone sensitive lipase (H.S.L.) of the foetuses, new borns and adult rats.
With the direct intraperitoneal administration of hPL to the foetuses in utero, the author could not find any remarkable change in the content of glucose, FFA, TG and serum T-N of the foetuses, even four hours after the injection.
Moreover, the activities of both lipases in foetuses were lower than those in newborns or adult rats.
From these experimental results, the following conclusion might be drawn : hPL secreted from the placenta affects primarily the maternal metabolism of carbohydrate and fat (glucose-fatty acid cycle), and induces hyperlipemia and hyperglycemia which influence the foetal metabolism via placenta.
The transported carbohydrate might be utilized as an energy source, but on the other hand, FFA might be utilized for synthesis of neutral fat of the foetus.
Furthermore, suspectedly, amino acids transported into the foetus through the placenta, might participate in protein synthesis in cooperation with hydrogen ion from glycolysis and actions of pituitary growth hormone, insuline etc.
In conclusion, hPL effects primarily the metabolism, especially glucose-fatty acid cycle of the maternal body, which supplies very important materials (FFA and glucose etc.) to the foetus for his anabolic process in growth.
Namely, the author may be able to emphasize that hPL is one of the very important “metabolism regulating factors” during pregnancy.

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