I. TSH secretion in patients with Graves' disease
1. In hyperthyroid patients, serum TSH levels determined by radioimmunoassay were undetectable (<1.0 μU/ml).
2. Three euthyroid patients with Graves' disease were treated with 1-methyl-2- mercaptoimidazole followed by thyroxine. Reciprocal changes in TSH and thyroxine levels were observed, but LATS did not change.
II. LATS in Graves' disease
1. Concentration and identification of LATS : LATS cantaining IgG which was separated from serum by DEAE-Sephadex column chromatography was passed through a CM-C column, and LATS fractions with high specific activity were obtained. The protein in these fractions with LATS was identified by immunoelectrophoresis as IgG.
There was no correlation between LATS activity and the concentrations of IgG, IgA and IgM which were measured by immunodiffusion technique.
2. LATS production by lymphocyte culture : Peripheral lymphocytes obtained from patients with Graves' disease were cultured with phytohemagglutinin, and LATS like activity was detected in the cultured medium. The LATS like activity was identified as LATS, by the time- and dose-response curves, association with IgG fraction of DEAE-Sephadex column chromatography, and neutralization with anti-IgG serum. Neither LATS nor TSH like activity was detected in the control cultured media.
3. The nature of LATS inhibitor in thyroid gland : LATS inhibitor was investigated in an attempt to find a specific antigen to LATS.
1) Incubation of LATS-IgG with human thyroid homogenates resulted in inhibition of LATS activity. When the subcellular fractions were used, LATS-inhibitory activity was demonstrated in microsomal fraction and cell sap, and dose-response relationship was obtained.
2) Following treatment with acidic buffer (pH 3.0) or 2M NaSCN where dissociation of antigen antibody complexes is known to occur, some of the LATS activity was recovered from microsomes which were previously incubated with LATS-IgG. Similar results were obtained when the mixture of the LATS-IgG and thyroid cell sap was treated with acidic buffer.
3) By incubation with thyroid cell sap, LATS activity was inhibited but TSH was not.
4) The LATS inhibitory activity of the cell sap was markedly reduced when the cell sap was pretreated with acidic buffer (pH 2.5, 3.0), 2M NaSCN or 6M urea.
5) The thyroid cell sap was fractionated by means of gel filtration using Sepharose 4B, but the LATS inhibitory activity was distributed to each fraction. When the cell sap was applied to a column of DEAE-cellulose, the LATS inhibitory activity was concentrated in the fractions which were eluted by the buffer with high ionic strength.
4. Relationship between LATS and antithyroid antibodies :
1) No correlation was observed between LATS activity and titer of antithyroid antibodies which were determined by TRC for thyroglobulin, CFT and cytotoxic factor assay with thyroid microsomes.
2) A case of “Graves' disease without hyperthyroidism” was presented. The patient has exophthalmos, goiter, LATS and antithyroid antibodies. Histological examination of thyroid gland revealed the findings of chronic thyroiditis.
5. No effect of LATS was found on adenyl cyclase activity which was determined by the method of Pastan but the further experiments are necessary to confirm the finding.

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It has been well known that hypothyroidism is caused by hypofunction of thyroid gland, in resulting in decrease of the level of circulating thyroid hormone. Since the function of thyroid gland is regulated by thyroid stimulating hormone (TSH) secreted from pituitary gland, hypofunction of the pituitary gland produces the hypothyroidism known well as secondary myxedema. In this type of myxedema, the production of thyroid hormone can be reduced by a defect in at least three steps of hormones biosynthesis, especially in term of thyroidal iodine metabolism as follow : (1) decrease of thyroidal radioiodine uptake (2) reduction of formation of both thyroxine (T4) and triiodothyronine (T3) (3) slowering the release of thyroid hormone from the gland into the blood. As well known, the facts are resulted by the reduction of TSH secretion from the pituitary gland. However, TSH level in the blood of primary myxedema is usually higher than that of secondary hypothyroidism. For examples, so-called idiopathic myxedema might have almost the same defect of thyroidal hormone synthetic step as that in the secondary hypothyroidism but TSH level in such patients can be over normal range due to negative feedback mechanism. Clinical pictures of patients with chronic thyroiditis suggested the presence of hypothyroidism but thyroidal radioiodine uptake of some of such patients showed within normal range because of over stimulation of thyroid by TSH. Since it has also well known that most of the primary hypothyroidism in the adult are followed by chronic thyroiditis, recently we studied the iodine metabolism in the thyroid of the primary myxedema and it was found that the myxedema could be caused by not only the atrophy of thyroid gland but also a defect of oxidation of iodide in the thyroid of chronic thyroiditis. The present report deals with our findings concerning abnormal radioiodine metabolism with mainly chromic thyroiditis.
Goiter KSCN-Test After I-Loading KSCN-Test BMR Uptake Goiter Idiopatic myxedema - np np dec dec - Myxedema due to chronic throdidstis + + np dec dec e radioiodine + - + dec dec c Masked hypothyroidism (A) - +or- + dec dec - (B) + - + dec dec c (c) + - - inc dec dec np indicates not performed ; - indicates no goiter or goiter was not present : + indicates presence of goiter : c indicates constant size of goiter : dec indicates decrease : inc indicates increase.
Clinical thyroid function's tests such as basal metabolic test (BMR), protein bound iodine in the serum (PBI), resin sponge T3 uptake (RSU) and thyroidal radioiodine 24 hr uptake (Uptake) were measured in all of patients who showed typical or subclinical signs of hypothyroidism. Then, thiocyanate test was employed to all patients with uptake more than 10% to know whether or not there might be present a defect of oxidation of iodide in the thyroid. Thereafter, iodinated lecithin containing 1 mg iodide administered to patients once daily for 14 days and again BMR, PBI, RSU and Uptake were measured (Iodine loading test). In a patient with negative thiocyanate test before iodine loading, this test was again performed after iodine loading. Primary myxedema except due to antithyroid drugs was divided into several groups according to both thiocyanate test and iodine loading test.
Clinical pictures suggested the presence of hypothyroidism but thyroid function's tests showed within normal range, in which some type of abnormal thyroidal radioiodine metabolism could be found by applying the thiocyanate and iodine loading tests. We called such patients as masked hypothyroidism.

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228 patients who had been proved histologically to have chronic thyroiditis were examined clinically, physicopathologically and immunologically.
1) Clinical finding : Among 228 patients, only 9 cases were male and the rest 219 cases were all female. The age of patients ranged 15 to 78 and most of them were observed at the age of 30-50. The goiter was the most common clinical sign and 45% of the patients had goiter alone without any other clinical signs. However, few patients had pain or tenderness of the thyroid, and complained palpitation, slight fever or easy fatigability as general symptoms. According to clinical judgement, 186 cases (81.5%) showed euthyroid state and 35 cases (15.4%) hypothyroid state. Of these 35 cases, 9 had no goiter, indicating “idiopathic myxedema” derived from chronic thyroiditis. 7 cases (3.1%) showed the temporary hyperthyroid state. 13 cases (5.7%) of chronic thyroiditis were complicated with several kinds of autoimmune disease, among which 7 cases of rheumatoid arthritis and 2 cases of thrombocytopenic purpura were observed. However the complications with the other autoimmune diesase such as Sjøgren's syndrome, myasthenia gravis, Addison's disease and chronic nephritis were rare and only one case of each disease was observed.
2) Thyroid histology : The needle biopsy of the thyroid was performed in all 228 cases. The findings were grouped into diffuse thyroiditis and focal thyroiditis according to Woolner's classification. 143 cases had diffuse thyroiditis and the rest 85 cases had focal thyroiditis.
3) Thyroid function test : In 18 cases of 66 patients with euthyroid state, PBI was higher than 7.5 μg%, and in 34 cases of 107 euthyroid patients, the 24 hour ¹³¹I uptake was higher than 40%. These data indicate that in patients with chronic thyroiditis PBI and ¹³¹I uptake does not necessarily represent the thyroid state of the patients. In contrast, T₃-resin sponge uptake corresponded well with the thyroid state of these patients. To the patients with chronic thyroiditis who had been treated with dessicated thyroid, 5 units of TSH (Armour) was administered on two succeeding days and ¹³¹I-uptake was measured after 1, 3, 6 and 24 hrs after the second injection. The degree of ¹³¹I-uptake was highest in patients with focal thyroiditis, lowest in those who had myxedema and moderate in those with diffuse thyroiditis. These data suggest that the degree of thyroid tissue damage corresponds well with the unresponsiveness to TSH. 4 hrs-Ca (15 mg/kg) infusions was performed in those patients who had been fully treated with T₄. The delay of Ca disappearance through ciruclation was most significant in those who had myxedema and mild in patients with diffuse thyroiditis. However, there was no difference between those with focal thyroiditis and normal control.
4) In the patients with chronic thyroiditis, 69% of them had an accelerated ESR, 41% had the elevated total serum protein and 19% had the low A/G. The elevation of zinc sulfate and thymol turbidity test were observed in about 60%. 71% of cases had the elevated plasma γ-globulin level (>1.58 g%) and especially the elevation of Ig-G was chracteristic. Some cases showed the elevated Ig-A or Ig-M levels also. In the patients with diffuse thyroiditis the thyroid autoantibodies were detected in 55% with a precipitation test (PT), in 92% with a, tanned red cell agglutination test (TRC) and in 77% with a complement fixation test (MCF). In contrast, in those who had focal thyroiditis, PT was positive in 8%, TRC in 62% and MCF in 56%. Thyroglobulin was an antigen for PT and TRC test, and microsome fraction was an antigen for MCF. Effects of TSH, LATS and sera of patients with chronic thyroiditis on the redox state of pyridine nucleotides in the thyroid tissue were studied with a specific microfluorometer.

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In the field of gynecology, estrogens have a very important function physiologically, and the assay of estrogen is indispensable for the study of gynecological disease.
However, estrogens are present in extremely low quantities in biological media which may contain large amounts of other substances with charactaristics similar to those of estrogens. The discovery of a large number of new estrogen metabolites in recent years has further complicated the problems.
As a test, despite the elegant methods of analysis for estrogenic steroids based on either column partitioning, countercurrent distribution or paper chromatography, there still remains a need for a more rapid and more sensitive assay procedure.
The application of gas liquid chromatography in quantitative analysis of steroids has brought about a new technique to the field of estrogen analysis. Gas liquid chromatography has a great advantage over conventional chromatography through its high sensitivity, speed and separation, simultaneous quantification and high resolution.
Wotiz and Marrian first applied gas liquid chromatography in quantitative analysis of estrogens in 1961. They attempted the gas chromatographic separation of fully acetylated derivatives of estrogens. In the same year, Luukkainen and Adlercreutz reported that trimethylsilyl ether was excellent in the quantitative determination of estrogens with gas liquid chromatography.
Since then, the quantitative assay of estrogens by gas liquid chromatography has made a remarkable progress through the improvement of detection apparatus, column packing or sample preparation.
Based on the report of Wotiz, I made modifications in trying to develop a method of determination of estrogens in urine of pregnant women with gas liquid chromatography.
The instrument was a Shimazu gas chromatograph, Model GC-IB. Columns were 225 cm x 4 mm i.d. packed with 100-140 mesh Gas Chrom P coated with SE 30. The columns were kept at 240°C, while the detector cell and flash heater were kept at 240°C and 290°C, respectively. The gas was nitrogen at 2.5 Kg/cm and 40 ml/min.
Estrogens were acetylated by dissolving them in a mixture of acetic anhydride and pyridine and keeping the tube at 37°C for 24 hours. Estrone acetate, estradiol diacetate, estriol triacetate were crystallized to a constant melting point and dissolved in ethanol for calibration standard. The approximate lower limit of detector of the three estrogens was 0.005μg. However, the relationship to the amount present was not completely linear, particularly at the value less than 0.05μg, but was sufficiently accurate at the value from 0.05μg to 1.5μg. On the same column under the same conditions, there were only minor changes in the calibration plots with time. The retention time (estrone acetate- 5.9 min, estradiol diacetate-8.3 min and estriol triacetate-14.1 min), did not change in the value more than 0.05μg. Therefore, the limit of useful sensitivity was 0.05μg of estrogen acetates. The area of the peak was obtained from the half- band width. Evidence for accuracy of the procedure was obtained through analysis of the estrogens following their addition to male urine sample. Mean value of recovery of estrone, estradiol and estriol was 65.0%, 73.1% and 75.1%, respectively.
In the quadruplicate determination of estrogens in urine or pregnant women, standard deviation of the three estrogens stayed within 10% of the mean value of measurement. Therefore, this method seemed to be adequately reproducible.
Using this method, various factors on estrogen determination so far conducted have been reinvestigated and the following results were obtained.
1) As to the method of hydrolysis, two-step hydrolysis gave the highest yield, but acid heating hydrolysis also gave equally favorable results.
2) The optimum duration of acid heating hydrolysis is 20 minutes.
3) Regarding an extracting solvent,

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Human chorionic gonadotropin (HCG) extracted by the Kaolin adsorption method from the urines of women in the 3rd and in the 10th month of pregnancy, and from the urine of patients with hydatidiform mole and with chorioepithelioma, respectively, were fractionated by gel filtration on Sephadex G-100, and their biological behaviors were critically examined. Various physicochemical analyses were carried out on HCG purified by DEAE-cellulose chromatography and gel filration on Sephadex G-100, and given an assay value of 10,000-12,000 IU/mg.
Gonadotropic activities were tested by the ovarian weight, the seminal vesicle weight and the augmentation test of Steelman-Pohley, using immature Wistar rats 21-24 days old and weighing 35-45 g. The protein content was determined by the adsorption at 280 mμ.
The sedimentation constant, the partial specific volume and the molecular weight of the purified HCG were determined. The molecular weight was obtained by the method of Archibald and the gel filtration method. Amino acids of the purified HCG were analyzed by an autoanalyzer, except for tryptophan which was determined by the method of Carpenter. Carbohydrates were subject to qualitative analysis, where neutral sugars were separated by the paper chromatography and subsequently identified by the thin layer chromatography, amino sugars were analyzed by the method of Belcher, and sialic acid by the method of Yamakawa. Neutral sugars were determined quantitatively as galactosemannose by the method of Makida, and fucose was determined by the method of Dische-Schettles. Amino sugars were determined as HCl-glucosamine by the method of Belcher, and sialic acid as N-acetylneuramic acid by the method of Aminoff.
In the gel filtration of the crude HCG, those from the urines of normal pregnant women and of patients with hydatidiform mole showed patterns with two active components, while HCG from the urine of patients with chorioepithelioma showed a pattern with a single peak and the retarded fraction was absent. The fact that HCG from the urines of the 3rd and the 10th month of pregnancy exhibit different patterns on refiltration is evidence that some differences exist in the two specimens.
The sedimentation constant of the purified HCG was found to be 2.74, the partial specific volume 0.74 and the molecular weight 35,900 by the method of Archibald. On the gel filtration method, two substances with different molecular weights were obtained. The molecular weights of the two compounds were found to be 103,000 and 35,500 by protein body (optical density) method, and 99,000 and 36,300 by the biological activity (ovarian weight) method. The agreement between the values obtained by the two different methods being satisfactory, and the ratio of the molecular weights of the two substances is about 3 to 1. The molecular weight of HCG has been considered to be about 30,000 usually. It has been verified that the presence of HCG of the larger molecular weight, has a value of about 100,000.
There exist 17 different amino acids in the purified HCG. The presence of methionine was not verified. The contents of the amino acids ranged from a maximum 7.6% for glutamic acid to a minimum of 1.0% for tryptophan. Different carbohydrates such as fucose, galactose, mannose, amino sugars, sialic acid were found. More quantitatively, the content of neutral sugars was found to be 12.5%, fucose 0.9%, amino sugars 9.3%, and sialic acid 11.3%.

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The examination of urinary 17-ketogenic steroids has been commonly used as the index of adrenocortical function. In the past fifteen years three different methods of 17-KGS determination were reported, which were the methods by Norymberski et al (1953), by Appleby et al (1955), and by Few (1961).
Each of twelve pure corticosteroids including the synthetic one was treated according to the three different 17-ketonizing reactions, which were bismuthate oxidation of Norymberski's method, bismuthate oxidation following borohydride reduction of Appleby's method, and metaperiodate oxidation following borohydride reduction of Few's method.
The 17-ketosteroids obtained as the products of these reactions with each corticosteroid were extracted, separated by paper chromatography, and examined by scanning of radioactivity and Zimmermann reaction on paper chromatogram. They were identified by their chromatographic behaviors, sulfuric acid chromogen spectra and infrared spectra.
The following results were obtained :
1) Zimmermann chromogen values of the products from each corticosteroid by three methods differed from each other. Generally, Zimmermann chromogen value obtained by bismuthate oxidation showed it was higher, on the contrary by metaperiodate oxidation following borohydride reduction it was lower. And epsecially this phenomenon was remarkable in the case of 11-keto-corticosteroids such as tetrahydrocortisone, cortisone and p rednisone.
2) Tetrahydrocortisone was converted into 11-keto-etiocholanolone by bismuthate oxidation, into 11-keto-etiocholanolone (25.2%) and 11β-hydroxy-etiocholanolone (68.1%) by bismuthate oxidation following borohydride reduction, and into 11β-hydroxy-etiocholanolone by metaperiodate oxidation following borohydride reduction. Tetrahydrocortisol was converted into 11-keto-etiocholanolone (33.5%) and 11β-hydroxy-etiocholanolone (66.5%) by bismuthate oxidation, and into the same products from tetrahydrocortisone by the other two methods. The 11-keto group was not itself chromogenic with Zimmermann reaction, but potentiated the chromogenic property of 17-ketosteroid; so, 11-ketoetiocholanolone was highly chromogenic with Zimmerman reaction compared to 11β-hydroxy-etiocholanolone.
3) Corticosteroids having double bond in ring A were converted into 17-ketosteroids by bismuthate oxidation, as well as cortisol into 11β-hydroxy-androst-4-ene-3, 17-dione, cortisone into androst-4-ene-3, 11, 17-trione, prednisolone into 11β-hydroxy-androst-1, 4-dien-3, 17-dione, and prednisone into androst-1, 4-diene-3, 11, 17-trione.
By bismuthate oxidation following borohydride reduction, cortisol and cortisone were converted into 3ξ, 11β-dihydroxy-androst-4-ene-17-one, 11β-hydroxy-androst-4-ene-3, 17-dione, androst-4-ene-3, 11, 17-trione and into an unidentified 17-KS; prednisolone and prednisone were converted into 3ξ-hydroxy-androst-1, 4, 9 (11) -triene-17-one and into five unidentified 17-KS's.
By metaperiodate oxidation following borohydride reduction, cortisol and cortisone were converted into 3α, β-hydroxy-isomers of 3ξ, 11β-dihydroxy-androst-4-ene-17-one; prednisolone and prednisone were converted into 3ξ, 11β-dihydroxy-androst-1, 4-diene-17-one and into 3ξ-hydroxy-androst-1, 4, 9 (11) -triene-17-one.
4) The products obtained from synthetic corticosteroids such as paramethasone, dexamethasone, betamethasone and triamcinolone had no chromogenicity with the Zimmermann reaction. Practically, the interference of these synthetic corticosteroids to urinary 17-KGS value during their administration to a patient with Addison's disease was negligible.
5) When 17-KGS determinations were done on a number of urines by these three methods, the values obtained by the Norymberski's method were consistently higher than those obtained by the other two methods.

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