A number of erythrocyte Na-K ATPase units were measured in 22 patients with hyperthyroid Graves' disease, 3 with primary hypothyroidism, 3 with simple obesity, 13 with chronic renal failure on hemodialysis, and 20 normal controls, using ouabain binding assay as described by DeLuise et al. The number of Na-K ATPase units, derived by maximal binding of³ H-ouabain, was decreased in patients with simple obesity (Mean ± SD, 0.26 ± 0.07pmol/10⁹ RBC), as compared with that in normal controls (0.39 ± 0.10), and a significant negative correlation between the number of the binding sites and the ratio of the measured body weight to the optimal body weight calculated by the modified Broca's method was observed in normal controls and patients with obesity (r= -0.51, p<0.05). The results agreed closely with that reported by DeLuise et al and provided validation of our estimates of the erythrocyte Na-K pump units. The maximal 3 H-ouabain binding was significantly diminished in patients with hyperthyroid Graves' disease (0.28 ± 0.07) when compared with that in normal controls, while the bindings were signifiantly elevated in patients with hypothyroidism (0.91 ± 0.26).
These results were in disagreement with those previously reported by animal studies where Na-K ATPase was found to be stimulated by thyroid hormones. It might be possible to partly explain this discrepancy by the degradation of Na-K ATPase in erythrocytes in addition to the apparent differences between erythrocytes and the other tissues and by the length of time that the tissue was exposed to the action of the hormones. Therefore, erythrocyte from normal controls and patients with hyperthyroid Graves' disease were divided into low and high density portions by a discontinuous 'percoll' density gradient centrifugation, and the bindings of the erythrocytes in two portions were separately measured. The bindings of erythrocyte in the higher density portion, representing relatively old-aged erythrocyte, were diminished to 92 ± 19% of the bindings of the original whole erythrocytes in normal controls. An even more marked reduction of the maximal bindings of ³ H-ouabain in old-aged erythrocytes was observed in patients with hyperthyroid Graves' disease (72 ± 26%). Moreover, this % reduction based on aging related significantly to serum T₄ concentrations in those patients (r = 0.85, p<0.05). These findings suggest that the number of erythrocyte Na-K ATPase units may reflect the overall peripheral metabolic state, regulated by thyroid hormone-dependent thermogenesis.
Therefore, to evaluate the peripheral metabolic status in patients with low T₃ syndrome, the maximal binding of ³ H-ouabain in erythrocyte was determined in patients with chronic renal failure on hemodialysis. Serum T₃ levels were less than 100 ng/dl in 9 of 13 patients, and most of them were considered to be low T₃ state. The bindings were significantly elevated in these patients (0.52 ± 0.13) when compared with those in normal controls (0.39 ± 0.10). Furthermore, there was a significantly negative correlation between the maximal binding and serum free T₄ level in these patients (r = -0.64, p<0.05).
To conclude, the maximal binding of ³ H-ouabain in erythrocyte could be used as the measure of peripheral thyroid hormone actions, and tissue hypothyroidism might exist in patients with chronic renal failure, most of whom have low circulating T₃ levels.

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5α-Androstane-3α, 17β-diol (A3αdiol) is a potent androgen, and is an end product of testosterone. Many authors measured A3αdiol levels in human plasma by various methods, but the levels of this steroid were very dissimilar. In order to validate such values, it was measured by gas chromatography - selected ion monitoring (GC-SIM) in this study.
A3αdiol, 5α-Androstane-3β, 17β-diol (A3βdiol) and Testosterone (T) in human peripheral serum were measured by GC-SIM at the same time. The TFA-derivatives of these compounds were analyzed after purification of the serum extract by Sephadex LH-20 microcolumn chromatography.
The sensitivity was good : (16.7pg/ml : A3αdiol, 26.7pg/ml : A3βdiol). The precision (CV = 2.75% : A3αdiol, 3.11% : A3βdiol) and the accuracy were better than ever reported.
Serum A3αdiol was measured in 131 healthy men aged 15-81 years and 5 healthy women aged 25-60 years. There were remarkable differences between individuals in the serum levels of A3αdiol, but the levels in male serum (>20y) showed a significant negative correlation with age (r=-0.560, p<0.01). When these healthy men were classified into three age groups of 20-39, 40-59 and 60-79 years, the values (mean ± SD) for serum A3αdiol were 189.3 ± 77.7 (n=20), 127.9 ± 59.5 (n=28), and 94.9 ± 52.9 (n=73) pg/ml, respectively. There were significant differences between the levels of this steroid in all age groups (p<0.01).
There was a weak but significant correlation between serum A3αdiol and T levels (r = 0.3235, p<0.01) in healthy men (25-77 years, n=77).
Determination of serum A3αdiol was influenced by age. The number of samples strongly influenced the decision of mean value of A3αdiol levels. These results suggested that these factors had to be made obvious when this steroid was studied.

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The significant influence of protein synthesis inhibitors on TRH-stimulated secretion of glycosylated TSH was investigated in the rat anterior pituitary in vitro. TRH stimulated secretion of (3H) glucosamine-labeled TSH but did not change incorporation of its labeled precursor into the pituitary TSH. Addition of cycloheximide significantly inhibited both incorporation of (14C) alanine into the pituitary protein and of (3H) glucosamine into the pituitary TSH even 3 hours after incubation, while it did not cause a significant change in secretion of glucosamine-labeled TSH. Apparent inhibition of (14C) alanine incorporation into the pituitary protein was produced by addition of actinomycin D in the 3 and 6 hour-incubation, but the same drug did not cause the significant change in the amount of (3H) glucosamine-labeled TSH in the anterior pituitary and medium. It is concluded from the present study that TRH plays a potential role in regulating carbohydrate synthesis of TSH prior to secretion, and messenger RNA is not essential for its role in the glycosylation of TSH.

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A radioimmunoassay for neurotensin (NT) and binding assay for NT receptor have been developed to determine neurotensin-like immunoreactivity (NTLI) concentration and to characterize NT receptor in the rat brain. The effect of various drugs affecting the dopaminergic system in the brain were also investigated to clarify the relationship between NT and dopamine.
NTLI was widely distributed in various regions of the rat brain : the highest level was 53.1 ± 14.1ng/g wet wt in the hypothalamus, 11.9 ± 6.0ng/g wet wt in the thalamus, 6.9 ± 0.7ng/g wet wt in the cerebral cortex and 0.7 ± 0.2ng/g wet wt in the cerebellar cortex.
The Bmax of NT receptor was 20.2 ± 4.6fmol/mg protein in the cerebral cortex, 16.0 ± 4.9fmol/mg protein in the hypothalamus, 13.3 ± 3.7fmol/mg protein in the thalamus and no detectable level in the cerebellar cortex. The Kd of NT receptor was 2.7 ± 0.7nM in the cerebral cortex, 1.9 ± 0.8nM in the hypothalamus, 1.1 ± 0.2nM in the thalamus.
Intraperitoneal (ip) bolus administration of L-DOPA caused the reduction in NTLI concentration from 9.2 ± 1.0ng/g wet wt to 5.3 ± 2.1ng/g wet wt and Bmax of NT receptor from 19.7 ± 3.1fmol/mg protein to 13.9 ± 2.6fmol/mg protein with no change in Kd. Intracerebroventricular administration of dopamine also induced a significant reduction of NTLI concentration and Bmax of NT receptor with no change in Kd. Inversely, ip bolus administration of α-methyldopa evoked an increase in NTLI concentration from 6.9 ± 0.9ng/g wet wt to 12.0 ± 3.0ng/g wet wt and that in Bmax of NT receptor from 15.9 ± 2.9fmol/mg protein to 24.2 ± 4.6fmol/mg protein. Serial administration of haloperidol evoked an increase in Bmax of NT receptor from 16.9 ± 1.5fmol/mg protein to 20.5 ± 1.9fmol/mg protein with no changes in Kd and NTLI concentration.
These results suggest that dopamine may modulate the action of NT in the rat cerabral cortex.

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Although 25-30 grams of calcium is transported into the fetus during pregnancy, it is suggested that the maternal bone might be kept at the same density as in non-pregnant women by measuring serum or urinary calcium concentrations and calcium regulating hormones simultaneously (Ohara et al. Folia Endocrinol., 1986).
In this study, the influence of pregnancy on the maternal bone was investigated by measuring the degree of bone density in the second metacarpal bone of pregnant or puerperal women in an X-ray picture using a microdensitometer and a computer (Microdensitometry method; MD method, Inoue et al., 1983).
Among six indices provided by this method, d (bone marrow width) tended to increase, but MCI (Barnett's metacarpal index) tended to decrease toward late pregnancy. GSmin, GSmax and ∑GS/D were significantly lower in the third trimester of pregnancy than in the second trimester of pregnancy. The densitometric patterns were A in most of the pregnant and puerperal women, but one case with the pattern of AB and two cases with the pattern of B were found in the third trimester of pregnancy. The sum of the scores of the six indices, which were based on the severity of each index, was within 0-3 in pregnant or puerperal women though it tended to increase as pregnancy progressed.
From these results, it was confirmed that maternal bone density was maintained within the normal limits throughout pregnancy and postpartum.

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The content and chemical and immunological properties of thyroglobulin (Tg) and the activity of thyroid peroxidase (TPO) were studied in nodular and extranodular (periphery) thyroid tissue from patients with functioning thyroid tumors (hot nodules).
From the present observations, the tumor extracts contained fairly large amounts of Tg, similar to that in the periphery tissue, while the content of Tg was markedly decreased in non-functioning thyroid tumors (cold nodules).
The iodine content of Tg was significantly higher in nodular tissue compared with periphery tissue. These observations were in contrast to the results seen in the cold nodules in which hormone synthesis was defective. On the nature of carbohydrate moiety as compared with normal tissue, oligosaccharides of Tg from the periphery tissues changed in a manner similar to that of tumor oligosaccharides. That is, high-mannose type oligosaccharides were markedly decreased whereas multiantennary and unidentified oligosaccharides were increased. These observations were similar to those of non-functioning tumors. The content of sialic acid decreased markedly both in the tumor and periphery tissues.
Immunologically, Tg preparations of the tumor and periphery tissue did not show the same affinity to the antibodies, depending on the content of iodine. Therefore, it seems that the contribution of the iodine content and iodo-amino acid has little or no significance in the heterogeneous nature of Tg-immunogenecity.
Benign tumors and the periphery tissue were subjected to assay for thyroid peroxidase activity by the method employing guaiacol as the second substrate. The specific activities of the tumors were generally higher than those of periphery tissues, both in functioning and non-functioning benign tumors. In non-functioning carcinoma, on the other hand, TPO activities were lower in tumor portions than in periphery portions. This indicates that non-functioning thyroid carcinomas have a different characteristic.

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The authors have developed a highly sensitive sandwich-type enzyme immunoassay (EIA) for serum thyroglobulin using the biotin-avidin system. The sensitivity of this EIA was 0.1ng/ml. The intra- and inter-assay coefficient of variation was 4-15% and 4-11%, respectively. Human Tg in serum was detectable in 100% of 63 normal subjects, and the normal range was from 2.3ng/ml to 47.4ng/ml. The recovery rate was very good.
Then we applied this EIA to the follow-up study of patients with thyroid cancer. Serum Tg levels were serially determined in 15 patients with thyroid cancer who had undergone thyroidectomy (n= 9 subtotal, n=6 total) and were receiving thyroid hormone suppression therapy. It was found that there were three groups in regard to the relationship between serum TSH and Tg. The first group showed normal TSH and normal Tg levels (n=4). The second group showed TSH levels that were lower than the lower limit of the assay, whereas Tg levels were within the normal limit (n = 5).
The last group showed both TSH and Tg that were lower than the lower limit (n=6). In one case in the last group, recurrence of thyroid cancer was forecast by the change of Tg levels (from below 0.1ng/ml to 5.9ng/ml). Therefore, it is important to suppress serum Tg and TSH levels as much as possible in order to obtain the best usefulness of serum Tg determination in the follow-up study of thyroid cancer in patients who have undergone subtotal thyroidectomy or have residual thyroid tissue.
We also measured Tg levels in patients with untreated Graves' disease. Twelve patients were negative for anti-Tg antibody and 18 were positive for anti-Tg antibody. Even in the antibody positive cases, serum Tg could be detected and their levels could be calculated from the recovery studies. The mean serum Tg levels in antibody positive cases were lower than in negative cases. These studies showed that our sensitive EIA for serum Tg determination was useful in practice in patients with thyroid diseases.

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In the present study, effects of angiotensin on the adrenal steroidogenesis were studied in essential hypertension, primary aldosteronism and renovascular hypertension (RVH).
Angiotensin III (A III), an analogue of angiotensin II, was administered to 17 normal volunteers (9 male and 8 female), 44 patients with essential hypertension (EH) (15 with high renin; HREH, 15 with normal renin; NREH and 14 with low renin; LREH), 8 patients with primary aldosteronism (5 with adrenal adenoma; APA and 3 with bilateral adrenocortical hyperplasia; IHA) and 5 patients with renovascular hypertension. In all the patients with hypertension and normal subjects, blood pressure (BP) and plasma concentrations of progesterone (P), corticosterone (B), aldosterone (Aldo), 17α-hydroxyprogesterone (17-OHP) and cortisol (F) were measured before and after intravenous administration of A III (0.1, 0.5, 1.0, 10, 20 and 40ng/kg/min, for 15 min, respectively).
1) BP rose from 164 ± 19/98 ± 8 to 180 ± 19/112 ± 10mmHg [systolic BP (SBP); P<0.01, diastolic BP (DBP); P<0.01] in HREH, from 162 ± 12/96 ± 7 to 186 ± 11/118 ± 8 mmHg in NREH (SBP; P<0.01, DBP; P<0.01), 165 ± 12/94 ± 8 to 202 ± 12/126 ± 9mmHg in LREH (SBP; P<0.001, P<0.001) and 118 ± 8/72 ± 7mmHg to 136 ± 11/88 ± 8mmHg in controls (SBP; P<0.01, DBP; P<0.01). The elevation in NREH and LREH was greater than that in HREH and controls. The elevations of BP both in APA and IHA were remarkably greater than that in controls and as similar as LREH (APA; 174 ± 21/103 ± 12 to 204 ± 18/ 136 ± 8mmHg, IHA; 176 ± 10/104 ± 4 to 206 ± 17/138 ± 10mmHg). The elevation in RVH was similar to that in NREH (173 ± 9/108 ± 8 to 194 ± 13/132 ± 10mmHg).
2) Plasma P increased from 25.5 ± 7.5 to 39.5 ± 13.8ng/100ml (P<0.001) in HREH, from 28.0 ± 7.7 to 45.3 ± 12.7ng/100ml (P<0.001) in NREH, from 23.8 ± 8.2 to 47.2 ± 19.4ng/ 100ml (P<0.001) in LREH and 26.6 ± 11.0 to 43.4 ± 14.6ng/100ml in controls. The increment in HREH or NREH was similar to that in controls (P<0.1, respectively), whereas greater than controls in LREH (P<0.05). Plasma P increased from 24.8 ± 6.6 to 37.5 ± 5.8 ng/100ml (P<0.001) in APA, from 46.3 ± 11.5 to 55.8 ± 18.6ng/100ml (P<0.2) in IHA and from 26.4 ± 3.8 to 39.7 ± 8.1ng/100ml (P<0.05) in RVH. The increment of P in APA was similar to that in controls, whereas smaller than controls in IHA and RVH. In one of 3 patients with IHA the increment was remarkably greater than that of controls (41.5 to 78.0ng/100ml).
3) Plasma B decreased in all groups of EH similarly in controls (HREH; from 290 ± 90 to 230 ± 100ng/100ml, NREH; 350 ± 110 to 250 ± 90ng/100ml, LREH; 290 ± 90 to 230 ± 100ng/100ml, normal; 340 ± 80 to 240 ± 80ng/100ml).
4) Plasma Aldo increased from 6.2 ± 2.0 to 8.2 ± 2.4ng/100ml (P<0.001) in HREH, from 6.1 ± 2.0 to 9.7 ± 4.1ng/100ml (P<0.001) in NREH, from 7.3 ± 1.9 to 15.9 ± 6.4ng/100ml (P<0.001) in LREH and from 6.4 ± 2.1 to 10.4 ± 2.7ng/100ml (P<0.001) in controls. The increment was smaller in HREH and greater in LREH than that in controls, whereas similar to that in NREH (P<0.05, P<0.02, P<0.1, respectively). Plasma Aldo did not increase in APA (28.0 ± 26.2 to 29.4 ± 25.7ng/100ml) and IHA (14.2 ± 3.3 to 27.4 ± 6.6ng/100ml) (P>0.1) whereas increased from 10.7 ± 5.4 to 24.9 ± 13.6ng/100ml in RVH, significantly (P<0.05). The increment in RVH was similar to that in LREH.

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