Several problems are involved in the studies on the biosynthesis of thyroid hormones : 1) How are iodine atoms incorporated into a thyroglobulin (TG) molecule? 2) How is the matured TG molecule built up? 3) how are carbohydrates incorporated in a TG molecule? 4) How are amino acids assembled to make up TG polypeptides? 5) How is TG hydrolyzed to produce T₃ and T₄? Recent progresses concerning the first four questions are briefly summarized and discussed below.
Tarutani's recent finding of non-iodinated TG from goitrogen-treated pigs confirmed that the most of iodine atoms are incorporated into TG after the formation of molecules with M.W. of 660,000. Micro villi or adjacent region was reported as a site of the iodination from biochemical and electron microscopic studies. With regard to the final step of the iodination, two DIT residues in TG polypeptides were suggested to be direct precursors of thyroxine on the basis of the comparison of chemically-iodinated and enzymatically iodinated TG with naturally existing TG. Studies on carbohydrate incorporation into TG were progressed by Spiro and Bouchilloux et. al. They concluded that carbohydrates are incorporated at microsomal membranes, presumablly at rough sufaced E.R, into TG polypeptides after the completion of peptide formation. Enzymological studies on the process also have been taken place by Spiro. The biochemical mechanism of thyroxine formation, the acutal process of molecular maturation in vivo, the roles of carbohydrate for the building of the molecule and for the biological functions of the molecule are some of the points remaining to be elucidated.
The in vitro studies on protein synthesis in the thyroid have been done by the author as well as American and French groups. Although Nunez reported that amino acids are incorporated into the same protein fraction as matured TG in his polysomal system, all others' results were not consistent with his conclusion and confirmed the microsomal synthesis of matured TG and the inability of polysomes in terms of production of the matured TG. In the author's studies a. new polysomal system was reconstructed which consisted of pig thyroid polysomes, liver t-RNA charged with labeled and unlabeled amino acids and liver cell sap as an enzyme mixture supplemented with the amino acid activating system. For the first 10 min. of incubation did the reaction proceed rapidly and slowed down after that period. An average length of the newly synthesized portion of the pep'tides in non-dialy-s able proteins which were released from polysomes by the treatment with RNase and EDTA (0.1 M) or with SDS (0.1 %), corresponded to 20 amino acid residues. Amino acid incorporation increased with the concentration of polysomes showing the dependency of the reaction on the polysomes. Six labeled amino acids were compared in their rates of incorporation into protein. Glutamic acid showed the highest value and others were in the same order as that of amino acid content of TG, though the order was not very different from that of a few other proteins including serum albumin. 20-30% of the labeled proteins released from polysomes were immunologically active against anti-TG serum when supllemented with anti-γ-globulin serum. The percentage of the active protein increased with reaction time slightly but reproduciblly even after total incorporation ceased its increase. Sizes of the labeled proteins were measured by a disc electrophoresis in the presence of 0.1 % SDS and were shown to be widely distributed from 20,000 to 150,000 as M.W. which was similar to a quarter subunit of TG.
These results strongly suggested that the thyroid polysomes could synthesize in vitro a considerable length of TG polypeptides in the quarter subunit of TG and the possibility to reconstruct a complete set of TG synthesizing system including an iodinating and a glycosylating system in addition to the peptide synthesizing system.

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Biochemical transformation of the thyroid hormone molecules in the mammalian tissue can occur at various points on the molecule. This is summarized as follows : (1) deiodination; (2) conjugation of glucuronic acid; (3) decarboxylation; and (4) deamination of the alanine side chain.
In our laboratories, the enzymes catalyzing reaction (4), oxidase and transaminase, have been purified from rat kidney.
The oxidase was obtained in crystal form from rat kidney mitochondria and soluble fractions and found to have 6 moles of FMN per mole of the enzyme (Molecular weight, 310,000). The enzyme catalyzes the oxidation of many α-amino monocarboxylic acids and α-hydroxy acids (L-configuration) including thyroid hormones. In recent study concerning intracellular distribution of the oxidase by sucrose density gradient centrifugation, the oxidase was found to be mainly located in microbodies (peroxisomes) rather than mitochondria.
The transaminase has been purified 30 fold from rat kidney mitochondrial extract. The enzyme requires pyridoxal-5'-phosphate as coenzyme and α-ketoglutarate as amino acceptor. The transaminase is highly specific for thyroid hormones and halogenated tyrosines. In contrast to tyrosine-α-ketoglutarate transaminase, this enzyme was not induced in vivo by hydrocortisone.
The pyruvic acid analogues of thyroid hormones have not been detected as product of the above transamination and oxidative deamination, while the corresponding acetic acid analogues were accumulated in the same condition. This may be attributable to the instability of the pyruvic acid analogues in alkaline media and chromatography in alkaline solvent systems.
On the basis of the fact that in the presence of aromatic α-keto acid reductase and NADH the main product of the deamination reactions is the lactic acid analogues of thyroid hormones, it can be concluded that the product of the first stage of the enzymatic deamination is the pyruvic acid analogues. Aromatic α-keto acid reductase is widely distributed in rat tissues and other animal tissues. This enzyme, purified from rat kidney; is highly specific for the aromatic keto acids corresponding to thyroid hormones and halogenated tyrosines.
Routes for side chain degradation of thyroid hormone (T₃, or T₄) are depicted as follows :
T₃ Oxidase Pyruvic acid analogue Transaminases NAD NADH+H⁺Lactic acid analogues Aromatic α-keto acid reductase Acetic acid analogues

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The action of a hormone to the organism can be generally considered in two fashions : the mechanism of its action and its physiological action. The mechanism of the action should include a primary event caused by the hormone in the target cell, followed by a series of successive reactions. The physiological action is composed of functions of the target organ, which are maintained or stimulated by the hormone sercreted or administered. In the strict sense, the mechanism of thyroid hormone action remains obscure though the physiological action implicates stimulations of their growths, maturations and basal metabolic rates, which indicate extensive changes in carbohydrate, lipid and protein metabolisms. The characteristic features of thyroid hormone action are as follows : first, the hormone affects almost all organs except brain, gonads and accessary reproductive organs, secondly, the hormone needs some length of time (the latent period) to exert the apparent action after its administration and thirdly, an optimal dose of the hormone is requeird to maintain the euthyroid state of the organism or to recover it to the normal level. Recently accumulated observations on the effects of minute amounts of thyroid hormone injected to hypothyroid animals were surveyed. Early and marked stimulations by the hormone administration are as follows : liver mitochondria respiration (State 4), synthesis of nuclear and cytoplasmic RNA (mainly r-RNA), RNA polymerase activity, synthesis of membrane phospholipid, incorporation of amino acid into protein by mitochondria and microsome and synthesis of respiratory enzymes which resulted in increased oxygen consumption.
In the connection with the first point, thyroid hormone may act on the target organs directly or indirectly because they affect each other (organ correlation). Among them, a reciprocal interrelationship (a feed back regulation) exists exceptionally between thyroid and adenohypophysis whose oxygen consumption was suppressed by thyroid hormone while stimulated after thyroidectomy. Since 1966, Tonoue and Yamamoto in this laboratory, have made a series of experiments to disclose the mechanism and found that thyroidectomy stimulated α-aminoisobutyric acid transport of rat adenohypophysis and 20 μg T₄ per 100 g body weight injected to thyroidectomized rats suppressed the augmented amino acid transport capacity. A similar finding was also made of experiments on ¹⁴ "C-alanine incorporation into the total protein of the adenohypophysis. Change in the ¹⁴C-alanine incorporation in the thyroid states was distinctly observed in TSH rich granules. The levels of free amino acids in rat adenohypophysis did not change or decrease with a few exception, that is, isoleucine, leucine and proline after thyroidectomy. Adenohypophyses from normal, thyroidectomized and thyroidectomized-and T₄ injected rats were incubated in KRB with ³'H-Phe and ¹⁴C-Arg. Triton X 100 (1%) extracts of the adenohypophyses were analysed with the use of disc electrophoresis. Thyroidectomy decreased markedly growth hormone and prolaction contents. While T₄ injection increased not only growth hormone and prolaction contents but also stimulated ³H-Phe and ¹⁴C-Arg incorporation rates into growth hormone and prolaction. The stimulations were clearly observable 12 hr after T₄ injection. Proteins separated between top of the gel and growth hormone area increased after thyroidectomy and T¹⁴ injection stimulated accumulation of the proteins. After thyroidectomy, marked changes were observed in carbohydrates metabolism of adenohypophysis, that is, increased output of ¹⁴CO₂ from 1-¹⁴C-glucose, decreased formation of lactic acid from glucose and increased glucose consumption followed by increase of leucine incorporation into the total protein.

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17-hydroxycorticosteroids, containing cortol compounds, were determined in human urine before and after loading with dexamethasone, ACTH-Z and metopirone administration. And they were also determined in urine of patients with various endocrine disorders.
To determine PorterSilber chromogens, thin layer chromatography and Porter-Silber reaction were mainly utilized, and to determine cortol compounds, periodic acid oxydation, alumina column chromatography and Zimmermann reaction were mainly employed.
In dexamethasone test, cortol compounds were more remarkably decreased than Porter-Silber chromogens in 9 of 10 cases. And in the 11-deoxy group they were decreased more remarkably than in the 11-oxy group, in 7 of 10 cases in Porter-Silber chromogens, and in all cases in cortol compounds.
In ACTH-Z test, they were more increased in the 11-oxy group than in the 11-deoxy group, in all cases in Porter-Silber chromogens, and 4 of 5 cases in cortol compounds.
In the ratio, cortol compounds/Porter-Silber chromogens, no peculiar pattern was observed. In metopirone test, the increase of 11-deoxy cortol was very charactaristic, practically 5.3-46.2 times as much as control, while the sum of 11-deoxy cortisol and 11-deoxy tetrahydrocortisol was 2.5-13.3 times. It was found to be more increased in the 11-deoxy group than in the 11-oxy group, as a matter of course.
As regards urinary 17-OHCS in patients with endocrine disorders, the excretion values are shown in table 7.
The main metabolic pathway of cortol compounds is the reduction of tetrahydrocompounds at C₂₀. However, it is reported that Reichsteins substance E or U is also one of the precursors of cortol compounds, and that its urinary excretion value is increased by ACTH administration. So from all these data, it is suggested that the decrease of urinary cortol compounds by dexamethasone administration is caused by the decrease of endogenous ACTH secretion. And also it is suggested that the increase of urinary cortol compounds by metopirone administration is caused by the increase of endogenous ACTH secretion.
As regards C₁₁ oxygenation, the decrease of urinary 11-oxy-17-OHCS in dexamethasone test should be due to the increase of endogenous ACTH secretion too.

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In an attempt to clarify the factors, especially the influence of steroid hormone on gonadotropin production in chorionic tissue, chorionic tissue was incubated in the media containing estrone, estradiol-17β, estriol or progesterone, respectively, and the change of gonadotropic activity, RNA and DNA content, RNase content and various phosphor fractions were examined.
Progesterone was found to inhibit the gonadotropin production in term placenta in vitro, but no inhibition was observed with estrogen. The presence of progesterone in incubation medium caused not only reduction of gonadotropin production but also a decrease of RNA content, increase of acid-soluble phosphor fraction and remarkable lowering of RNA/DNA ratio.
These changes are identical with the results of incubation experiment of chorionic tissue in the medium containing RNase. In other words, progesterone apparently activates RNase in chorionic tissue of the term placenta, which reduces RNA content and causes the reduction of gonadotropin production. Therefore, it is very probable that progesterone (steroid hormone) controls the production of gonadotropin (proteohormone) through RNA metabolism, and that an interregulating mechanism might possibly exist between steroidogenesis and production of proteohormone in chorionic tissue.

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It is generally considered that the antigen which is used for radioimmunoassay should be highly purified. We have developed a method for sensitive, precise, and specific radioimmunoassay of luteinizing hormone (LH) in human blood using partially purified human chorionic gonadotropin (HCG) as the antigen.
Based on the well known cross-reactivity of human LH with HCG, antisera were developed against commercial crude HCG (GONATROPIN, 2,000-3,000 I.U./mg, Teikokuzoki Co. Tokyo) in guinea pigs. The antisera thus obtained were absorbed both with pediatric urine powder prepared by Albert's method and with 6M. urea-treated human menopausal gonadotropin (Schmidt-Elmendorf et al, J. Endocrin, 24 : 153, 1962), (Fig. 1). These procedures were carried out for the absorption of possibly contaminated antibodies against non-specific protein of urine and FSH. One of these antisera was selected for this sensitive and specific radioimmunoassay of LH. Partially purified HCG (5,000 I.U./mg, Teikokuzoki Co.) without further purification was used for the iodination with ¹³¹I and for the standard. This HCG had the immunological potency of 8,500 U of 2nd International Reference Preparation of Human Menopausal Gonadotropin (2-IRP HMG) per milligram. The method of Hunter, Greenwood and Glover was used for the iodination of HCG, and ¹³¹I-HCG was purified by gelfiltration with Sephadex G 75.
0.1 cc of 5% bovine serum albumin (BSA) in phosphate buffer (0.05 M, pH 7.4), 0.2 cc of standards or samples, 0.1cc of 1 : 20,000 diluted anti-HCG serum and 0.1cc of ¹³¹I-HCG (about 0.1 mμg) were added into small test tubes. The mixed solutions were incubated for 4 or 5 days at 4°C, followed by the addition of 0.2 cc of normal guinea pig serum (1 : 200) and 0.2 cc of appropriately diluted rabbit antiguinea pig γ-globulin antiserum. The diluent for standards, anti HCG serum, ¹³¹I-HCG, normal guinea pig, and anti-guinea pig serum was 0.5% BSA in phosphate buffer (0.05 M, pH 7.4).
Dose-response curves for the standard HCG, 2-IRP HMG, pregnant woman's serum, and postmenopausal woman's serum were all identical (Fig. 5 and 6). Porcine ACTH, human growth hormone, and human TSH did not cross-react significantly in this assay system (Fig. 4). LH levels in plasma or sera from 15 adult normal men, 10 normal women (in their follicular phase), 10 postmenopasual women, and 10 panhypopituitary patients ranged as follows : 9.3-33.0, 4.2-28.8, 39-131, and 0-9.3 mU of 2-IRP HMG per cc, respectively. All the values above well agreed with those reported previously from other laboratories (Table 1).
Although the HCG we used was not highly purified, this assay system proved to be valid. The impurity (ies) contaminated in our HCG might be immunologically very feeble and/or the absorption procedure performed on anti-HCG serum might make up for a drawback of the antigen.

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It is a generally recognised postulation that secretion of ACTH from the pituitary takes place as a response of organism to the stressor.
However, the mechanism of ACTH secretion in this case is not yet quite clearly analysed. There are several postulations, such as regulation through feedback system based upon the possible reduction of plasma steroid level caused by the increased utilization of steroid in peripheral tissue (Sayers & Sayers). This postulation was later discussed and questioned by Yates (1961, 1967), Smelik (1963) and Hodges (1963).
These authors are of the opinion that steroid level in the plasma does not play any role in the ACTH secretion in the case of stress. Yates (1967) proposed to divide the stress into two types, i.e., corticoid sensitive and corticoid insensitive stress. The experimental studies used by him were of the type to investigate the effect of extra administered corticoid, not of the fluctuation of plasma steroid level in situ. Therefore, it is necessary to investigate directly the plasma ACTH activity and corticoid level simultaneously in the case of stress to clarify the stiuation.
For us it became possible to investigate the plasma ACTH activity as a routine method through a bioassay system using the steroid output of in vitro perfused beef adrenocortical slices as parameter of the ACTH activity of the test material (Yasui-Folia Endocri. Jap., 41 : 643, 1965).
Thus, a series of experiments were planned to investigate the fluctuation of plasma ACTH activity and corticoid level in rats placed under various kinds of stresses. Throughout the whole experiment female rats weighing 120 to 150 gm. were used as test animals. As stress the following procedures were used : 1). Ether vapour exposure for one minute, 2). Synthetic Lysine-8-Vasopressin (Sandoz) 100 mU./100 gm. b.w. i.v. hereafter abr. as L-8-V., 3), Histamine 250μg./100gm. b.w. i.v., 4). Bell-alarming (94 earhorn for 5 seconds then one second intermission), 5). Skin incision longitudinal 5 cm. long on the abdominal skin, 6). Laparotomy longitudinal 5 cm. long with exposure of intestine.
In the case of repetition of stimuli various combinations of these stresses were adopted. As expression of plasma ACTH activity fig./gm./ml. was used, which means the amount of steroid excreted per ml. of perfusion medium per gm. beef adrenocortical slices per 1.0 ml, plasma sample.
All experimental procedures were carried out from 9.00 to 12.00 A.M. under consideration of diurnal fluctuation of pituitary-adrenocortical activity.
As anaesthesia 5 mg. of pentobarbital-Na per 100 gm. body weight was given intraperitonially. Blood was taken after decapitation under heparination.
The results are summarised as follows :
1). Under pentobarbital anesthesia plasma ACTH activity and corticosterone level decrease gradually to reach the minimum value at 45 minutes after the commencement of anesthesia and at the interval between 90 to 120 minutes these two values return to the starting level. Responses, of these two values upon laparotomy and L-8-V were most significant at 45 minutes after anesthesia.
2). Successive determination of plasma ACTH activity after giving various stimuli disclosed the existence of three different patterns of ACTH release according to the sort and loading methods of stimuli.
As the first pattern plasma ACTH shows abrupt increase and returns very soon to the starting level as in the case of L-8-V, i.v., histamine i.v., and ether vapour exposure. In the second pattern the plasma ACTH activity reaches abruptly its peak value and stays on an elevated level up to a certain time interval as in the case of bell ringing and skin incision.
In the third pattern plasma ACTH activity reaches its peak abruptly and stays on this elevated level for long time, as in the case of laparotomy.
Throughout three kinds of patterns,

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It is well known that the liver plays an important role in steroid hormone metabolism, and there are many reports concerning the disturbance of corticoid metabolism in hepatic disease. While some investigators suggested the hypofunction of adrenalcortex in liver diseases from the autopsy findings, few reports using stimulation test on adrenalcortex have been presented.
This study was designed to make clear the adrenalcortical function in hepatic patients by ACTH-Z stimulation.
Normal adults (Control), patients in the convalescent stage of acute hepatitis (A group), patients with chronic hepatitis (B group) and with cirrhosis of the liver (C group) were subjected. ACTH-Z (20 U) was injected intramuscularly at 9 a.m. for two days and adrenocortical response was observed by the assay of urinary total and free 17-OHCS and plasma 17-OHCS.
1) Prestimulatory levels of 17-OHCS in hepatic patients. Urinary total 17-OHCS was decreased in all groups compared with normal control. Regarding urinary free 17-OHCS, increased excretion was observed in C group but in the other groups no significant difference was seen as compared with control. Plasma 17-OHCS levels in each group were almost the same as normal control. From these basic levels, it is impossible to discuss about the aderonocortical function, and they rather suggest the abnormality of corticoid metabolism in hepatic diseases.
2) Adrenocortical response to ACTH-Z stimulation in hepatic patients. Urinary total 17-OHCS variation : In A group there were two different patterns. One was low and another high but a late response pattern. In B group adrenocortical response was nearly the same as normal control. But C group showed low response pattern. Urinary free 17-OHCS variation : Very high but late response was observed in A group and low response in B and C. Plasma 17-OHCS variation : Same maximum response levels appeared 4 to 8 hours after administration of ACTH-Z each day in the Control group. But there was a significant difference in hepatic patients. In A, B and C groups the maximum level on the first day of stimulation was lower than normal. However, on the second day, A group showed the same maximum response level as Control, and B and C groups showed lower level than that on the first day. From these results it is presumed that there is hypofunction of adrenalcortex besides the disturbance of steroid metabolism in hepatic diseases especially in chronic hepatitis and cirrhosis of the liver.

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In order to elucidate possible influences of certain hormones on the activity of hexokinase (HK) and glucokinase (GK) which play important roles in the initial step of sugar utilization, effect of fasting on the activity of these enzymes in rat liver, effect of insulin on the activity in the liver of alloxan-diabetic rats and also influences of varied hormonal environments on the activity in steroid-diabetic rats were investigated.
1. Markedly lowered level of the GK activity in rat liver as the result of fasting was restored almost to the normal level upon refeeding. The HK activity also tended to be decreased by fasting, and showed a tendency toward restoration after refeeding.
2. Remarkable fall in the GK activity was also observed in alloxan-diabetic animals which was restored almost to the normal level by insulin treatment for 48 hours. The HK activity also showed a similar tendency, though not being significant statistically.
3. In the liver of steroid-diabetic rats under the condition employed in the present study, the GK activity level proved to be higher than normal, and when insulin was administered to these animals, the level was increased furthermore.
4. The liver GK activity of adrenalectomized rats was significantly lower than normal and it was recovered nearly to the normal level by cortisol treatment.
5. Cortisol administration to alloxan-diabetic animals did not bring any increase in the enzyme activity, but, on the contrary, caused further decrease.
6. Upon alloxan treatment of rats followed by adrenalectomy, the liver GK activity was decreased notably, and when insulin was administered to these animals, restoration of the activity was observed. The extent of this increase was not influenced significantly by an administration of 10 mg of cortisol in addition to insulin.
7. From all these results, a close relationship between insulin and the activity of the enzymes under examination was revealed and the increased liver GK activity observed on the steroid-diabetic rats under the present experimental condition was presumed to be due to the intervention of insulin oversecretion. Cortisol is considered not to inhibit the GK-inducing effect of insulin.

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It was reported by the present author in the preceding paper that the increase in liver glucokinase (GK) activity in steroid-diabetic rats is presumed to arise through an oversecretion of insulin and also that cortisol does not inhibit the GK-inducing effect of insulin. The present work was undertaken to investigate possible effects of hypophysectomy, thyroidectomy, and thyroxine, ACTH and growth hormone administration on the GK and hexokinase (HK) activity of rat liver, and also to know whether or not administration of each of these hormones yields an antagonizing action on the GK-inducing effect of insulin.
1. Upon administration of thyroxine to normal rats, the liver HK activity was increased significantly, but the GK activity remained unchanged.
2. Following thyroidectomy, the GK activity showed a significant decrease, while the HK activity remained unaltered. When T₄ was administered to such ectomized animals, the GK activity showed a tendency to restoration.
3. Growth hormone administration to normal rats did not bring any significant change in the activity of these two enzymes.
4. In the liver of hypophysectomized rats, the GK activity was significantly lower than normal. After growth hormone or ACTH administration to these animals, no tendency to the recovery of this enzyme activity was observed.
5. The decreased level of the liver GK activity in alloxan-diabetic animals was raised almost to the normal by insulin treatment, but simultaneous administration of T₄ or of growth hormone together with insulin did not bring any enhancement in the increase. This result may indicate the fact that neither T₄ nor growth hormone blocks the GK-inducing effect of insulin.

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