During acid hydrolysis the urinary oestrogens are partly destroyed. Enzymatic hydrolysis usually does not destroy oestrgens, but it results generally in relatively incomplete cleavage of conjugates, since inhibitory substances present in the urine interfere with the hydrolytic action of the enzymes. The principles of gel filtration were applied to conjuated oestrogens and a procedure was developed (by C.G. Beling) for the purification and concentration of conjugated oestrogens in the urine and the removal of urinary inhibitors on the enzymic hydrolysis of oestrogens.
This idea led to the present investigation and resulted in the development of a 2Step hydrolysis of urinary conjugated oestrogens. By this method the oestrogen excretions were studied during normal menstrual cycle, pregnancy and endocrine disorders.

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Biological activities of 17α-acetoxy-6-chloro-pregna-4, 6-diene-3, 20-dione (chlormadinone acetate ; CMA) were investigated in comparison with those of 17α-acetoxy-6α-methyl-pregnane-3, 20-dione (medroxyprogesterone acetate ; MAP). CMA produced marked progestational activity either subcutaneously or orally in the uterine carbonic anhydrase test, and its relative potency is approximately 30 times the activity of progesterone in the subcutaneous assay and about 40 times the activity of norethisterone in the oral assay. CMA also possessed more activity than MAP in oral route. Deciduomatogenic activities of these two steroids subcutaneously injected into the mice were approximately the same, and their relative potencies against progesterone were approximately 4. Anti-gonadotropic activity of CMA was almost equal to that of progesterone administered subcutaneously either in the subcutaneous or in the oral test with parabiotic mice. MAP, however, produced marked activity of gonadotropin inhibition in the subcutaneous test but was as active as CMA in the oral route. In the ACTH-inhibition test in rats, CMA was less active than MAP either by subcutaneous or by oral route. Other hormonal activities such as androgenic, myogenic, estrogenic or anti-inflammatory were not manifested by the CMA treatment. Both CMA and MAP had no anesthetic activity in mice. These data suggest that CMA is a potent orally active progestational substance with a negligible activity to suppress pituitary ACTH and gonadotropin secretion.

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We examined the method of computing urinary 17KGS in accordance with the Appleby-Norymberski method (1955), improved on the method of excluding imporitities in the urinary extract, and confirmed definitely that various corticoids were converted 17KS.
By applying this modified Appleby-Norymberski method clinically, we obtained new interesting knowledge.
Results are as follows :
(1) Secrosterone (6α-21-dimethy1-17α-ethinyl-testosterone) and testosterone are not transformed into 17KS, consequently, estimation values are not affected by them.
(2) Average amounts of urinary 17KGS excretion of 32 amenorrhoic females and 32 normal females were 5.7 mg and 4.5 mg, respectively. Values of amenorrhoic females were usually in the higher part of normal range.
(3) Metabolism of corticoids was accelerated in the latter half of the menstration cycle.
(4) Levels of urinary 17KGS of pregnancy are considerable above the normal range and that in the first trimester of pregnancy are in high rate, but in comparatively low rate in the latter half, because metabolism of steroids is inhibited by estrogen at that time.
(5) In cases which were administered with 17α-OH-progesterone Capronate, urinary total 17KGS levels increased. But 17KS and its fraction were not changed remarkably. Urinary 17KGS decreased with the administration E.P. (17α-ethynyl-testosterone ; ethinyl estradiol), and had no change with the administration of secrosterone and 0.5 mg Dexamethazone and less.
This method is useful for the clinical examinations of subclinical adrenal disorder and hypermetabolism which has not been found by the Poter-Silber method.

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Cellular thyroxine-binding protein is considered to participate in the cellular penetration as well as in the cellular metabolism of the thyroid hormones. It has been demonstrated in rat and rabbit skeletal muscle and brain extracts, but not detected in the liver which is known to play an important role in the metabolism of the hormones. The present report is concerned with demonstrating specific cellular proteins which are capable of binding the thyroid hormones in the liver, by using paper electrophoresis.
The liver soluble proteins, to which ¹³¹I-labeled thyroid hormones were bound in vitro and in vivo, were fractionated by conventional and reverse-flow paper electrophoresis, and the evidence for the presence of both cellular thyroxine-binding protein and cellular triiodothyronine-binding protein in rat and human liver was obtained. The cellular thyroxine-binding protein from rat liver had a mobility intermediate between α₂- and β-globulin in conventional electrophoresis, while it had the mobility of α-globulin in reverse-flow electrophoresis. Almost the same results were obtained in human liver. The celluar triiodothyronine-binding protein from rat liver had the mobility of β-globulin in conventional electrophoresis, while it had a mobility intermediate between α- and β-globulin in reverse-flow electrophoresis. Similar results were obtained in human liver.
The cellular thyroxine-binding protein from rat skeletal muscle, which seemed to bind triiodothyronine, had a mobility intermediate between β- and γ-globulin in conventional electrophoresis, while it had the mobility of α-globulin in reverse-flow electrophoresis.
The serum thyroxine-binding protein of the rat, which seemed to bind triiodothyronine very loosely, had a mobility similar to that of albumin in conventional electrophoresis, wile it had a mobility intermediate between albumin and α-globulin in reverse-flow electrophoresis.

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A previous report described the existence of the cellular thyroxine-binding protein (C-T₄ BP) in rat and human liver, and strongly suggested the possibility of the existence of cellular triiodothyronine-binding protein (C-T₃BP) which might be different from C-T₄BP in these tissues. The present report deals with column chromatographic studies on C-T₄BP and C-T₃BP.
Rat serum and liver soluble proteins, to which ¹³¹I-labeled L-thyroxine (T₄-¹³¹I) and L-triiodothyronine (T₃-¹³¹I) were bound in vitro and in vivo, were fractionated by DEAE-cellulose chromatography. Thyroxine-¹³¹I bound to liver soluble proteins showed two peaks. The minor peak, emerging in the earlier stage of elution, corresponded to serum T₄BP in chromatographies of serum and of a mixture of serum and liver soluble proteins containing T₄-¹³¹I. Small amounts of free T₄-¹³¹I were eluted in the abscence of protein under similar conditions, but showed a different elution pattern. Then, it could be concluded that the major peak represented C-T₄BP which was different from serum T₄BP. This C-T₄BP showed a lower affinity for T₄ than that of serum T₄BP in dialysis studies.
Triiodothyronine-¹³¹I bound to liver soluble proteins showed a single peak, which was eluted in a different area from those of C-T₄BP and serum T₃BP (the same as serum T₄BP). The chromatography of a mixture of serum and liver soluble proteins containing T₃-¹³¹I also supported the conclusion that this radioactive peak repesented C-T₃BP which was different from serum T₃BP. Further, the C-T₃BP which was found to contain T₃-¹³¹I only was clearly separated from C-T₄BP in chromatography of liver soluble proteins obtained from the rat injected with equal amounts of T₄-¹³¹I and T₃-¹³¹I. The affinity of C-T₃BP for T₃ was shown to decrease more markedly than that of C-T₄BP for T₄ as the pH of the medium was decreased from 8 to 6. These studies provide the confirmatory evidence for the existence of C-T₃BP which is different from C-T₄BP, and indicate the possibility of the purification of C-T₄BP and C-T₃BP by this procedure.

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