The radioreceptor assay system for TSH is considered to be useful in quantitating the hormone and analyzing the mechanism of its action. The assay was established, and the interaction of abnormal thyroid stimulators in Graves' patients was evaluated in this assay system.
A 10,000xg fraction of human thyroid homogenates was used as the receptor. Human TSH supplied from NIH was iodinated by using lactoperoxidase. The binding of ¹²⁵I-TSH to the receptor was small, and ¹²⁵I-TSH was further purified by the receptor binding. The receptor (25mg equivalent), purified ¹²⁵I-TSH, and standard TSH or a sample were incubated at 37°C for 60 min in a final volume of 300/μl. The binding of ¹²⁵I-TSH to the receptor was time- and temperature-dependent with optimal binding under the conditions described above.
The binding was completely inhibited by the addition of human, bovine and ovine TSH and partially inhibited by high concentrations of HCG, FSH-LH. However, there was no cross reactivity with insulin, prostaglandin E₁, E₂, T³, T⁴ and NaI. The assay was sensitive enough to detect 5 to 50μU of TSH. The amount of TSH bound to the receptor was almost parallel to the TSH concentration which is necessary to stimulate human thyroid adenylate cyclase activity.
Studies of dissociation kinetics and Scatchard plot indicated that there were two classes of TSH receptors in the human thyroid. A higher association constant was calculated as 1.5 × 10⁸M^-1.
LATS-IgG from a patient with Graves' disease completely inhibited the binding of ¹²⁵I-TSH to the receptor, and studies of Lineweaver-Burk plot suggested that TSH and LATS-IgG shared common binding sites.
The radioreceptor assay of TSH appears to be useful in evaluating the abnormal thyroid stimulators present in Graves' disease.

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In the radioreceptor assay system for TSH, serum immunoglobulin G (IgG) from some patients with Graves' disease has been shown to inhibit the binding of labelled TSH to its receptor sites. In order to clarify the properties of these TSH-binding inhibitor immunoglobulins (TBII) in patients with Graves' disease, TBII were measured in sera from 31 untreated and 51 ¹³¹I-treated patients, and their relation to clinical and laboratory findings was studied. TBII were detected in 18 (60%) out of 31 patients with untreated Graves' disease. TBII levels in these patients correlated well with thyroidal ^99mTc uptake at 30 min and also with the grade of epithelial hyperplasia of thyroid follicles. There was no significant correlation between TBII and serum T₃, serum T₄, free T₄ index, antibody titers against thyroglobulin and microsomes, or association of exophthalmos.
There were many patients with Graves' disease whose sera contained high TBII levels but no detectable bioassayable thyroid-stimulating activity (LATS), and in these patients a close correlation was observed between serum levels of TBII and bioassayable LATS-protector activity.
In patients with Graves' disease who had been treated by ¹³¹I from 5 to 17 years before, the incidence of TBII was very low at 20% (10/51). All except two cases having TBII were found to be still thyrotoxic. Thus, TBII were detected in 8 out of 10 thyrotoxic patients and in only 2 out of 18 euthyroid and none of 23 hypothyroid patients.
These findings suggest that TBII in patients with Graves' disease were in close association with human thyroid stimulating activity, and that TBII might be useful as an indicator for checking the effectiveness of the treatment.

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TSH-binding inhibitor immunoglobulins (TBII) have been detected not only in patients with Graves' disease but also in those with Hashimoto's thyroiditis by using the radioreceptor assay of TSH. In the present study, the properties of TBII in patients with Hashimoto's thyroiditis are discussed.
Two (7%) of 29 patients with Hashimoto's thyroiditis had detectable levels of TBII in their γ-globulin fractions. Both patients were untreated and clinically hypothyroid. One of them had no goiter, and her thyroidal ^99mTc uptake was 0% (normal range : 0.4-3.0%). Despite having potent TSH-binding inhibitor activity in the TSH radioreceptor assay, her serum or its IgG fraction (H-IgG) did not contain any significant anti-TSH antibody, LATS, LATS-protector or human thyroid adenylate cyclase (AC) stimulating activity. This H-IgG inhibited both human thyroid AC stimulation and c-AMP increase in mice thyroid glands induced by TSH or LATS. Furthermore, her serum caused significant inhibition of ¹³¹I-release by LATS in a McKenzie mouse bioassay. The present study demonstrates that the serum of one patient with Hashimoto's thyroiditis contained antibodies which 1) blocked the binding of labelled TSH to the receptor, 2) had no thyroid-stimulating activity by themselves, and 3) inhibited AC stimulation by TSH. Such antibodies may cause unresponsiveness to TSH stimulation, hypothyroidism, and, if this state persists for a long time, eventually may result in atrophy of the thyroid tissue. Further, these data indicate that TBII detected by the TSH radioreceptor assay did not always show thyroid stimulating activities.

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As the greater part of the immunoreactive angiotensin II in cerebrospinal fluid has been suggested to be angiotensin III, a comparison was made between the effects on vasopressin release of angiotensin II and angiotensin III administered into the third cerebral ventricle in conscious male rats. The blood samples were collected 90 seconds after the injection of angiotensin II or angiotensin III by means of decapitation.
Plasma vasopressin (μU/ml) extracted and determined by radioimmunoassay were 2.3±0.8, 6.7±5.0, 14.0±2.2, 16.3±4.3 and 20.7±2.5 (mean ± SEM), respectively following the injection of 0, 10, 25, 50 and 10Ong of angiotensin II. The increases in plasma vasopressin produced by angiotensin II 25, 50 and 10Ong were statistically significant (p<0.05). On the other hand, plasma vasopressin following the injection of 22.7 and 45.4ng of angiotensin III, which are equimolar to 25 and 50ng of angiotensin II each, were 14.9±2.7 and 16.3±5.6, respectively. No significant difference was found between the effect on plasma vasopressin of angiotensin II and that of angiotensin III at the dose level of 24.3 or 48.6 p.mol.
These data indicate that angiotensin III is equipotent to angiotensin II in terms of vasopressin release when administered into the third cerebral ventricle. The possible role of angiotensin III in the brain on vasopressin secretion is discussed.

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The high pressure liquid chromatographic (HPLC) technique was developed to separate and quantitate the synthetic corticosteroids (s-CS) which are widely used clinically. 1) 12 kinds of s-CS in alcoholic solvent and 2) some of their metabolites in the plasma and urine of healthy subjects with oral administration of s-CS were investigated for the preliminary work.
The results are summarized as follows :
1) Cortisol sodium phosphate, Dexamethasone 21, disodium phosphate, Paramethasone acetate, Cortisol acetate, Cortisone acetate, Methylprednisolone acetate, Prednisone, Dexamethasone, 9α-fluorocortisol, Betamethasone, Triamcinolone, and Prednisolone in ethanol were clearly separated by HPLC from Cortisol (F). In the suitable condition of the HPLC (LC-2 type) with a Zorbax SIL column, organic solvent (cyclohexane : dichloromethane : ethanol = 9 : 4 : 1) -carrier mobile phases and UV detector, the retention time of each s-CS was obviously different from that of F. The calibration curve was obtained in a linear line with regards to each s-CS. The mean recovery was 97.6% and the coefficient of variation were 1.6 (intraassay) and 7.2 (interassay) %. The sensitivity of the steroid determination was 200pg order.
2) The serial changes in plasma concentrations of s-CS; CS-metabolites and endogenous F were shown in 3 healthy males and 2 females following oral administration of the s-CS. The separated metabolites in number and quality depended on the kind of s-CS. Prednisone and other kinds of the acidified products were separated from prednisolone in the plasma and urinary samples of the healthy subjects as well as Addisonian patients.
In conclusion, the HPLC method is useful for the separation and quantitation of the UV-absorbing CS of human plasma and urine. The obtained chromatograms may be an indication of the metabolic state of the subject with treatment of s-CS.

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To obtain an in vitro method of LATS assay which is equally sensitive to a McKenzie bioassay, we compared the quality of cAMP generation-stimulation, thyroidal adenyl cyclase stimulation, and the stimulation of T³-release from mouse thyroid slices. Among those indicators of thyroid stimulation, stimulation by IgG of the release of T³ from incubated thyroid slices is the most sensitive, reproducible and reliable indicator, reflecting faithfully the results of a McKenzie bioassay. Stimulation of cAMP generation in slice manifested less sensitivity, but reasonable reproducibility. However, adenyl cyclase stimulation by IgG of patients with Graves' disease was erratic in two successive experiments, probably due to a non-specific effect of IgG. When adenyl cyclase stimulation was adopted as a sole indicator of thyroid stimulation, the results must be interpreted cautiously.

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Though prostaglandin (PG) derivatives are widely used for the induction of labor, the physiological levels in plasma are still controversial because of the difficulty concerning its measurement.
Since the main urinary metabolite of PG-F2_α (PG-2_α MUM) has been identified as 5α, 7α-dihydroxy-11-ketotetranoprosta-1, 16-dioic acid by Granström et al., the measurement of this metabolite has been developed by using Gas-chromatography-mass-spectrome try or radioimmunoassay.
This paper reports the examination of a radioimmunoassay for PG-2_α MUM and further, the changes of this metabolite during pregnancy, labor and the early postpartum period by using this assay method.
In this assay system, ¹²⁵I was used for the labelling isotope, and the double antibody technique was used for the separation of bound and free PG-2_α MUM.
Any extraction and purification procedures were unnecessary.
The range of measurement (0.1-10ng/ml) was sensitive enough for the measurement of urinary levels, and some dilution of urinary samples were required.
The values of intra-assay coefficient of variation (CV) obtained from quintriplicate measurements were 7.2-14.3%.
The values of inter-assay CV obtained from five different runs were less than 15.0%.
Thus, together with the mean recovery rate (94.8%), this assay method provides sufficient precision and accuracy for clinical purposes.
By using this method the following results were obtained :
PG-2_α MUM levels during normal pregnancy were examined using 218 samples of urine taken during 24 hours. At the first and second trimesters, PG-F2_α MUM levels were lower than at non-pregnant levels (18.73±5.75ng/ml), which were obtained from 11 healthy nonpregnant women. At the third trimester, PG-F2_α MUM levels increased to the levels of 1.5 times higher than those of the first or second trimester.
Serial measurements in 12 normal pregnant women from the 8th month of pregnancy to delivery revealed that PG-F2_α MUM levels remained rather steady during pregnancy but increased rapidly during labor.
The maximum level (250.27±36.33ng/ml) was attained at 2 hours after delivery, and the elevated level returned to that before delivery at the second day of postpartum.
The present results suggest that PG-F2_α MUM levels increased as the result of uterine contraction.

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In order to clarify the regulation of aldosterone secretion in low-renin essential hypertension, plasma renin activity and plasma aldosterone were measured before and after an upright position with a furosemide administration in 168 cases of essential hypertension and 5 cases of primary aldosteronism. Essential hypertension was divided into low-, normal- and high-renin groups from the results obtained by this renin stimulating experiment. Plasma cortisol and plasma ACTH were measured before and after the experiment in 40 cases of essential hypertension and in 5 cases of primary aldosteronism. The effects of angiotensin II and ACTH-Z on plasma aldosterone and circulating plasma volume were measured and evaluated using the radio iodine labeled serum albumin method.
In the low-renin group, the upright position with a furosemide administration induced an elevation in plasma aldosterone without a significant change in PRA. However, plasma aldosterone correlated positively with PRA even in the low-renin group (before r=0.507, after r=0.759), but did not correlate with ACTH before and after the experiment. On the other hand, in primary aldosteronism, plasma aldosterone correlated positively with plasma cortisol and ACTH, but it did not correlate with PRA during the experiment. The increase of plasma aldosterone during the angiotensin II administration was lower in the low-renin group and in primary aldosteronism than it was in the normal- or high-renin groups. The increment of plasma aldosterone after ACTH-Z was similar in all groups; however, it was significantly higher in primary aldosteronism than it was in essential hypertension. The circulating plasma volume was similar in all groups except that it was high in primary aldosteronism.
The data indicate that in low-renin essential hypertension, the adrenal receptor is not necessarily sensitive to angiotensin II, and plasma aldosterone change is regulated by the renin-angiotensin system. The syndrome of documented primary mineralocorticoid excess is rare and constitutes a small fraction of low-renin essential hypertension. On the other hand, in primary aldosteronism, the adrenal receptor is not sensitive to angiotensin II, and plasma aldosterone is regulated by an endogeneous ACTH secretion rather than the renin-angiotensin system.

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In a case of dexamethasone responsive mineralocorticoid excess, we found a peculiar phenomenon; namely, that the administration of cortisol or cortisone in a replacement dose produced apparent hypokalemia even under dexamethasone suppression.
The clinical course of this Japanese girl is described in Table 1. At the age of three years, a laparotomy was performed under the diagnosis of Conn's syndrome, but no adrenal tumor was found on either side, and subtotal adrenalectomy was done bilaterally. Microscopic findings of both adrenals showed a disarrangement of adrenocortical zonation, and the initial stage of adrenal hyperplasia was suspected. One month after surgery, hypertension and hypokalemia reappeared. The administration of dexamethasone was dramatically effective in suppressing the symptoms of mineralocorticoid excess. At the age of 13 years, sexual maturation and ovarial estrogen secretion were normal and a regular menstrual cycle was observed. From these clinical pictures and the results of steroid secretion studies, the 17-alpha-hydroxylase deficiency syndrome was ruled out. In an attempt to alter the therapeutic agent from dexamethasone to natural glucocorticoid, it was found that a single injection of cortisone acetate (25mg) produced a hypertensive crisis and the administration of cortisol 30mg daily for six days produced apparent hypokalemia (Fig. 1). From these findings, some abnormalities of cortisol metabolism or hypersensitiveness to cortisol with regard to mineralocorticoid activity were suggested.
Tracer doses of cortisol-1.2-³H and corticosterone-4-¹⁴C were injected simultaneously, and the urinary metabolites were analyzed on successive 24-hour urine collections. After ethyl acetate washing to remove free steroids, washed urine was subjected to beta-glucuronidase hydrolysis, and liberated steroids were extracted by dichloromethane. The extract was purified by solvents partition and paper chromatography. Finally tetrahydrometabolites of cortisol and corticosterone were isolated by the Bush B-5 and modified B-5 systems respectively. Each paper strip was scanned using a gas-flow strip scanner, and a radioactinogram was obtained.
The results were as follows :
1. In this patient, the peak of tetrahydrocortisone (THE) was not observed, while a relative increase of allotetrahydrocortisol (allo-THF, 5a-THF) was observed (Fig. 4).
2. The major peak of corticosterone metabolite was located less polarly than tetrahydrocorticosterone (THB). This substance was suspected to be allotetrahydrocorticosterone (allo-THB, 5_α-THB) from its RF value. The peak corresponding to 11-dehydro-tetrahydrocorticosterone (THA) was not observed clearly (Fig. 5).
3. After a three day administration of Zinc-corticotropin (Cortrosyn-z) this pattern of metabolism was unaltered (Fig. 6 & 7).
From these results, it is very likely that this patient suffers from abnormalities of steroid metabolism especially on reduced 11-beta-hydroxy-dehydrogenation and elevated 5-alpha-reduction. The pattern of metabolic abnormality resembled closely cortisol 11-beta-ketoreductase deficiency (11-beta-hydroxy-dehydrogenase deficiency) which was recently reported by New and collaborators. (Pediat. Research 12 : 416, 1978)
But the clinical pictures are somewhat different. In their case amelioration of the symptoms of mineralocorticoid excess was introduced by salt restriction rather than by dexamethasone.

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