Triiodothyronine methyl ester hydrochloride (T₃·MEH) was obtained by acid methanol method of Ashley and Harington. T₃·MEH was conjugated to bovine serum albumin (BSA) by carbodiimide method of Oliver et al. 3.2 mg of T₃·MEH-BSA complex was dissolved into 0.8 ml of sterile physiological saline solution. This solution was emulsified in equivalent volume of complete Freund's adjuvant and injected into toe-pads of rabbits (0.4 ml per injection). Rabbits were boosted with intramuscular injections of 1 mg of antigen in complete adjuvant every month. Sera were obtained one week after the last injection one year later.
a) T₃ in 0.1 ml of unknown serum or standard T3 added to the serum of cretinism was extracted with 2 ml of methanol. Extraction rate was 85±2%. After evaporation of methanol, extracted T₃ was dissolved into 1.0 ml of barbital buffer (pH 8.6, 0.05 M) containing BSA in 0.5%.
b) Because of binding of the ¹²⁵I-T₃ to serum protein, direct assay of serum yielded high nondisease related T₃ values. In order to inhibit such binding, 0.01 ml of dinitrophenol (DNP, 1.20 x 10^-3 M) or diphenylhydantoin (DPH, 5.85 x 10^-3 M) was added to 0.1 ml of crude serum diluted with 0.89 ml of BSA diluent.
To each assay system in a) or b), 0.1 ml of ¹²⁵I-T₃, in BSA diluent (less than 25 pg, specific activity 70-450 μCi/μg, Abbott or Mallinckrodt) and 0.1 ml of antiserum diluted with BSA diluent (1 : 1,000-2,000) were added. This gave a total volume of 1.2 ml in each assay system. The mixtures were incubated at 5°C for 18 hours. Then 0.2 ml of dextran-coated charcoal (Charcoal 5.0 g and dextran 0.5 g in 400 ml of water) was added to them. One hour after shaking at room temperature for 5 min., mixtures were contrifuged at 3,000 r.p.m. at 5°C for 15 min. and then radioactivities of supernatant and precipitate were counted in a well-typed scintillation counter. Percentage of bound ¹²⁵I-T₃ was calculated.
The ability of various T₃ analogues to inhibit the binding of ¹²⁵I-T₃, to antibody was compared to that of T₃ (1.00) with the following results : l-thyroxine, 0.0021; triiodothyroformic acid, 0.066; triiodothyropropionic acid, 0.081; triiodothyroacetic acid, 0.061; tetraiodothyroformic acid, 0.00014; tetraiodothyroacetic acid, 0.0013; l-monoiodotyrosine, 0.000070 and l-diiodotyrosine, 0.000059, respectively.
Mean T₃, values in methanol extraction and DPH addition method in euthyroid, hyperthyroid and hypothyroid patients were 111±19 and 126±48,490±160 and 440± 179, and 46±15 and 41 ±24 ng/dl (SD), respectively. In vitro addition of 20,000 ng/dl of l-T₄ to normal serum (serum T₄, 10.5 μg/dl) resulted in no change of T₃ concentration. The dilution curve of serum T₃ in a hyperthyroid patient (serum T₃, 1,600 ng/dl) was pallalel to the standard curve.

View full abstract

Resting plasma TSH values in normal subjects and patients with various endocrine disorders were estimated by radioimmunoassay with the double antibody technique, using Human Thyrotropin Research Standard A as a standard.
The following results were obtained :
1) The values in 36 normal adults (ranging from 16 to 38 in age) were distributed, between undetectable (<1.7 μU/ml) and 3.6 μU/ml, and two thirds of them were undetectable.
2) Values in 17 aged subjects (over 70 years old) ranged from <1.7 to 8.9 μUlml. Undetectable values were found only in 5 cases of them.
3) In most cases of 53 patients with untreated hyperthyroidism, TSH values were undetectable, but in 5 cases detectable. In a group of patients during antithyroid drug administration, in which relative high serum PBI levels (>5.0 μg/dl) were observed, the TSH values were also undetectable, except in a few cases. But, in another group (PBI <5.0 μg/dl), about half the number of patients had detectable values.
4) Values in 21 patients with primary hypothyroidism ranged from 6.6 μU/ml to 1155 μU/ml.
5) In 3 cases of pituitary chromophobe adenoma with secondary hypothyroidism, undetectable values were observed.
6) In other cases of pituitary chromophobe adenoma and craniophrayngioma the values were roughly normal, and the values in patients with acromegaly were also similar, but in patients with diabetes insipidus they were slightly elevated.
7) 8 patients with simple goiter had values ranging from <1.7 μU/ml to 6.6 μU/ml. Only 2 of them had undetectable values.

View full abstract

The purpose of this study is to set up the optimum schedule of pituitary reserve function test by loading synthetic LH-releasing hormone (LH-RH) and to define the range of normal response in men, and women at different stages of the menstrual cycle, and to compare these results with response in the patients with apparent pituitary dysfunction.
Preparation of LH-RH synthetized by a classical method was diluted with 10 ml of saline and injected into volunteers intravenously in 30 sec to 1 min. The venous blood was drawn before and 15 min, 30 min, 60 min, 120 min, and 180 min after the injection of LH-RH. Serum levels of LH and FSH were determined by a double antibody radio-immunoassay (RIA) using Calbiochem RIA kit, and 2nd IRP-HMG was used as the standard. The minimal detectable dose was 0.5 mIU/ml for LH assay and 1.0 mIU/ ml for FSH assay. The subjects for this test were 5 normal males, 8 normal menstruating women each at early follicular phase, preovulatory phase and luteal phase, a prepubertal girl, a climacteric woman, 3 postmenopausal women, 7 patients with Sheehan's syndrome and a chromophobe adenoma patient.
LH release from the pituitary was observed by administration of as little as 5 pg of LH-RH and the dose-response relationship was found up to 400 μg between injected LH-RH and the maximum increase of serum LH in a normal male. By intravenous injection of 100 μg of LH-RH as a test dose, serum LH showed the peak usually 30 min after the injection then declined sharply, while serum FSH level in most cases reached a peak 60 min after the stimulation then decreased more gradually than LH.
The mean value of both gonadotropins in normal male increased significantly to 69.2±12.6 mIU/ml (9.1 fold increase) in LH and 13.2±2.0 mIU/ml (1.9 fold) in FSH, and it was apparent that the increase in LH was more remarkable than in FSH. The maximum responses (mean±SE) of serum LH during the normal menstrual cycle were 279.4±87.6 mIU/ml (6.7 fold) in the preovulatory phase, 48.2±3.5 mIU/ml (5.6 fold) in the luteal phase, 29.9±4.9 (2.8 fold) in the early follicular phase. While maximum increase of serum FSH reached 26.7±11.9 mIU/ml (3.0 fold) in the preovulatory phase, 18.5±3.6 mIU/ml (1.7 fold) in the early follicular phase and 7.0±1.7 mIU/ml (1.8 fold) in the luteal phase. The most striking increase in both LH and FSH was observed at the preovulatory stage. Results of LH-RH test in relation to age revealed positive responses in a 7-year-old girl, a climacteric woman and post-menopausal women. Three cases out of 7 cases of Sheehan's syndrome showed low response to LH-RH and the remaining 4 cases and a case of pituitary tumor had almost no response.
From the above data, it is concluded that intravenous injection of 100 μg of synthetic LH-RH followed by a determination of serum level of LH and FSH by RIA, is useful to clarify the pituitary reserve function especially of gonadotropin secretion.

View full abstract

Two antibody radioimmunoassay (RIA) for human TSH was studied using human TSH RIA set from Calbiochem. The assay was carried out with the pre-precipitation technique described by Hales and Randle for insulin. Purified human TSH (5-7 USP Units/ mg, LH ca 100 IU/mg) was iodinated with ¹²⁵I. Human Thyrotropin Research Standard A (HTSH R-STD-A) and Human Pituitary Thyrotropic Hormone 68/38 were kindly supplied by the WHO International Laboratories for Biological Standards, for use as standard. The results were expressed as μU of HTSH R-STD-A per ml of serum.
Some characteristics of the radioimmunoassay was examined, before serum TSH levels in subjects of various endocrine states were measured. As the anti-TSH serum used in this study considerably reacted with human pituitary LH (LER 960) and HCG (Fig. 1), a dose of HCG which had to be added to each incubation tube to eliminate the cross-reaction was investigated. When 20 IU of HCG was added to each tube, a complete dose response curve for standard human TSH was still demonstrable (Fig. 2). However, 10 IU of HCG in each tube was enough to eliminate the effects of HCG as much as 100 IU/ml (Fig. 3) and LH as much as 200 ng (LER 960) /ml (Fig. 4) in the assay. The dose response curves of sera from three euthyroid and one hypothyroid subjects were identical with the standard curve (Fig. 5). Antiserum to human TSH was used in a final dilution of 1 : 60,000, antiserum to rabbit γ-globulin in a final dilution of 1. : 240. 10 IU of HCG was added to each tube. The standard HTSH was dissolved in a buffer with serum from a completely hypophysectomized patient (Fig. 2).
No remarkable alteration of serum TSH was observed during a normal menstrual cycle in twelve healthy women (Fig. 6). Age-dependent changes of serum TSH were investigated in 205 women and 145 men. Comparatively low levels of serum TSH were observed in infants and subjects at old age of both sexes. In general, serum TSH levels in males were slightly lower than t hose in females throughout their lives (Figs. 7 and 8). Serum TSH levels in girls at the time of menarche were slightly but significantly (p<0.01) higher than the levels in all females at ages other than menarche (Fig. 7). In males, such a elevation of serum TSH at menarche was not observed.
Serum TSH levels of the patients who failed to ovulate with gonadotropins were slightly lower than those of patients who ovulated with gonadotropins. In particular, the serum TSH level of the patients who failed to ovulate with human menopausal gonadotropin and human chorionic gonadotropin (HCG) was significantly lower than the level of those who ovulated with pregnant mare serum gonadotropin and HCG (p<0.05) (Fig. 9).

View full abstract

Serum TSH levels after thyrotropin releasing factor (TRF) administration were estimated by radioimmunoassay in aged men to demonstrate whether the endocrine system, especially the function of pituitary TSH secretion, would be affected by the aging process or not.
To compare the effects of TRF on plasma TSH levels, 50 to 100 μg synthetic TRF injections were performed intravenously in 10 cases of aged men over seventy and in 11 cases of young euthyroid men of the third decade.
The results were as follows.
1) In the aged men group there was a raise in plasma TSH to peak levels at 15 minutes after TRF injection and thereafter a fall toward basal levels.
2) The changes on plasma TSH levels after TRF administration in the aged men were almost the same as those obtained in young normal subjects.
3) Therefore, it was susggeted that pituitary TSH reserve to definite doses of TRF in aged men were maintained much the same as those in young normal subjects.

View full abstract

To investigate the change of corticosteroidogenesis under an environmental stress, rats were exposed to mechanical vibration (vertically, 2G with 10 cycle/sec.) for 5 hours. The results obtained are as follow.
1. The level of corticosterone in adrenal and serum increased significantly (by analysis of variance, P<0.01) about 3 times compared with control.
2. The level of adrenal NADPH and NADH increased significantly (by analysis of variance, P<0.05) but only about 1.3 times compared with control. There was not a change of NADP or NAD level in adrenal.
3. Swelling of adrenal mitochondria from stressed rat, which was estimated as a change with Ca⁺⁺ at 520 mμ absorbance, resulted in a marked decrease (by Welch's test, P<0.01). It was suggested that there was some change of mitochondria membrane in adrenal.
The findings mentioned above were observed soon after the beginning of vibration and continuously during vibration.
As a result, it is considered that NADPH, coenzyme for steroid hydroxyration, is increased a little but the turnover rate of NADP system may be enhanced by a change of mitochondrial membrane.

View full abstract

In order to clarify the pathophysiological significance of the sympathetic nervous system in thyroid disorders, attempts have been made to determine the urinary excretion of catecholamine in the clinical course of patients with thyroid disorders. Furthermore, in addition to urinary excretion of catecholamine, the myocardial concentration of catecholamine and monoamine oxidase activity in myocardium were observed in rabbits with thyroid disorders induced by thyroid hormone or surgical procedure.
1) In 24-hour urine, adrenaline and noradrenaline have been found 9.3±0.4 μg/ day and 25.0±1.1 μg/day in the normal subjects, 14.0±0.8, μg/day and 27.1±1.1 μg/ day in hyperthyroidism without exophthalmus, 20.1±1.1 μg/day and 46.1±2.9 μg/ day in those with exophthalmus and 9.3±1.2 μg/day and 29.5±4.2 μg/day in hypothyroidism. (P<0.001 against normal value. All values are shown as meani±SEM.)
2) Positive correlation was found between urinary excretion of adrenaline and PBI or Triosorb test in hyperthyroid patients with exophthalmus.
3) Urinary excretion of catecholamine was reduced along with the clinical improvement during the treatment with methimazole in hyperthyroid patients.
4) Increasing enhancement in urinary excretion of catecholamine was found in triiodothyronine-treated rabbits during the course of treatment, but no significant change in thyroidectomized rabbits.
5) The myocardial concentration of catecholamine was significantly decreased in triiodothyronine-treated rabbits, while increased in thyroidectomized rabbits.
6) A comparison of the urinary excretion of noradrenaline with the myocardial concentration of noradrenaline revealed an inverse relationship.
7) The monoamine oxidase activity in myocardium was found to be increased in triiodothyronine-treated rabbits but no change in thyroidectomized rabbits.
As the results of these investigation, it is presumed that the hyperthyroidism is associated with hyperactivity of the sympathetic nervous system, reflected in increased urinary excretion of catecholamine associated with a deficit of myocardial concentration of catecholamine and an increased monoamine oxidase activity in myocardium.

View full abstract