To study the significance of TRH in the hypothalamo-pituitary-thyroid axis measurement of TRH in body fluid are needed. we previously reported TRH radioimmunoassay for urine.
TRH radioimmunoaassay for serum has not established yet, because TRH immunoreactivity is inactivated with serum.
We investigated effects of various factors on this inactivation and method for prevention of this inactivation.
Synthetic TRH was added to normal human serum at 4°C and incubated at 60°C, 37°C, 20°C, 4°C or -20°C for various intervals. After incubation, recovery of TRH was measured.
After one hour incubation, recovery of TRH was 9. 2% at 37°C, 34. 5% at 20°C, 100% at 4°C or -20°C. Incubation of TRH serum mixtures at 65°C after incubation at 37°C resulted in some recovery of TRH.
After one hour incubation at 37°C, recovery of TRH was 9. 2% at serum pH 7. 0, 100% at serum pH 3. 0 to 5. 0 or 11.0.
Recovery of TRH was increased in accordance with stepwisely increase of serum dilution. Concentrations of serum thyroid hormone did not affect recovery of TRH. Smaller quantities of TRH were more rapidly inactivated.
Inactivation of TRH immunoreactivity could be prevented addition of BAL (over 0.25 mg/ml) or mixture of 8-Hydroxyquinoline (HQ) and Tween 20 (T) (over 0. 1 mg/ml of HQ and 11mg/ml of T). Duration of effectiveness of BAL was short. Effectiveness of HQT continued for 12 weeks, if HQT treated serum was stored at -20°C.
From above data it was suggested that TRH immunoreactivity might be inactivated with enzyme system and other factors and TRH levels in the serum might be able to measure with addition of HQT to serum.

抄録全体を表示

Growth hormone (GH) responses to L-dopa, 2-Br-α-ergocryptine (CB-154), thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hormone (LH-RH), glucagon and glucose were investigated in six patients with active acromegaly. The following results were obtained.
1) Subcutaneous injection of 1 mg glucagon caused a clear-cut decrease in plasma GH levels in 5 out of 6 active acromegalic patients at 30 minutes after the injection. In 2 out of 6 patients a rebound of plasma GH was observed.
2) In three out of six patients with active acromegaly, oral administration of 0. 5 g L-dopa caused a significant suppression of plasma GH levels.
3) CB-154 (2. 5 mg) administered orally elicited a marked decrease in plasma G11 levels in the same three patients who showed a significant suppressive GH response to L-dopa, and the inhibitory effect of CB-154 on GH secretion lasted for 6 hours. These patients who had a GH response to L-dopa or CB-154 were named “responders”.
4) Intravenous administration of TRH resulted in a significant increase in plasma GH in 4 patients 3 of whom were responders and the other a non-responder.
5) Pretreatment with CB-154 did not modify the TRH-induced GH increase in all patients who had a positive response to TRH.
6) A significant increase in plasma GH was elicited by the intravenous injection of 100 μg LH-RH in 3 out of 6 patients with acromegaly.
7) When oral administration of CB-154 had been given 2 hours before LH-RH, the GH response to LH-RH was blunted in two of three patients who had a LH-RH-induced increase in plasma GH levels.

抄録全体を表示

The major role of the corpus luteum is biosynthesis of progesterone. Luteal function has been investigated by following plasma progesterone concentrations and by studying ultrastructural and histochemical changes in corpora lutea. Recently, changes in enzyme activities concerned with formation and degradation of progesterone are taken into investigation in order to understand the regulation of luteal function. In rat ovaries, progestational potency of ovarian secretions has been shown to be regulated by the activity of 20α-hydroxysteroid dehydrogenase (20α-HSD), which catabolizes progesterone to 20α-hydroxypregn-4-en-3-one, progestationally inert steroid. In regressing corpora lutea, extensive conversion of progesterone to 20α-hydroxypregn-4-en-3-one occurred with a marked increase in 20α-HSD activity as well as a decrease in plasma progesterone concentrations. On the other hand, histochemical studies of glucose-6-phosphate dehydrogenase (G 6 PDH) and Δ₅-3β-hydroxysteroid dehydrogenase (3β-HSD) have been investigated without any remarkable changes in corpora lutea at their early stages of luteolysis. In the present study the activities of steroidogenic enzymes in corpora lutea of pregnant rats are measured after treatment with a variety of abortifacient drugs, and compared with those in corpora lutea of 1 day post partum rats which showed changes characteristic of spontaneous luteolysis.
On days 7 to 9 of pregnancy, Wistar-strain pregnant rats were injected with either prostaglandin F_2α (PGF_2α), aminoglutethimide or clomiphene citrate (clomid). Animals were sacrificed 15 to 63 hrs. after the last injection, and implantation sites were inspected. Ovaries were removed, and corpora lutea dissected free, weighed and homogenized. The homogenate was centrifuged at 105,000 g for 60 min. The supernatant solution was assayed for the activities of G 6 PDH, 6-phosphogluconate dehydrogenase (6 PGDH), malic enzyme, ATP citrate lyase, 20α-HSD and pyruvate kinase. The pellet fraction was re-homogenized, and centrifuged at 2,000 g for 5 min. The supernatant solution was used for the assay of 3β-HSD.
Complete fetal resorption was observed in all rats treated with PGF_2α, while 7 out of 15 rats (47%) treated with both PGF_2α and LH-RH maitained pregnancy. In intact rats after treatment with both drugs, lutein cells showed ultrastructures characteristic for luteolysis, although the degree of luteolysis was greatly diminished compared with PGF_2α-treated ones. In agreement with these ultrastructural findings, 20α-HSD activity in corpora lutea was maintained at a rather low level in intact rats, while it was increased moderately in aborted ones after treatment with both drugs. In PGF_2α-treated rats, G 6 PDH activity increased to 140% and malic enzyme activity decreased to 27% of the activity in control rats.
In aminoglutethimide-treated rats, the activities of G 6 PDH and malic enzyme were decreased, while 20α-HSD activity was maintained at a low level. When the dose level was increased, complete abortion was observed. In corpora lutea of these rats 20α-HSD was activated moderately and G 6 PDH had a slightly higher activity than control ones, whereas malic enzyme activity fell to much lower level. These changes of enzyme activities were quite similar to those observed in PGF_2α-treated ones.
In rats treated with clomid the activities of those enzymes concerned with steroidogenesis, G 6 PDH, malic enzyme and ATP citrate lyase decreased moderately as compared with control rats, while 20α-HSD activity was maintained at a very low level. 6 PGDH, 3β-HSD and pyruvate kinase activities were not altered appreciably in these rats. The activities of 20α-HSD in corpora lutea of 1 day post partum rats,

抄録全体を表示

The present report deals with the measurement of serum thyroglobulin (Tg) in various thyroid disorders by using radioimmunoassay (RIA). The results were as follows :
1) A specific and simple solid-state RIA for the measurement of Tg in human serum was used. This system was a direct RIA using plastic cups coated with crude anti-thyroglobulin antibodies (anti-Tg) and ¹²⁵I labeled purified anti-Tg. The purification of anti-Tg was performed by affinity chromatography using Tg-Sepharose, as an immunoadsorbent.
2) Affinity chromatography was carried out using a modification of the method of Cuatrecasas. The immunoadsorbent (Tg-Sepharose conjugate) was used in a column procedure for the isolation of anti-Tg from globulin fractions obtained from Hashimoto's sera. The elution was performed with 4M NaI or 0.17 M Glycine-HCl with pH 2. 3. The eluted materials contained a very small amount of Tg which was removed by Sephadex G-200 chromatography using the same elution buffer. The high purity of the anti-Tg obtained was demonstrated by the fact that almost all of the final product was bound with Tg by using Sephadex G-200 chromatography.
3) A direct RIA consisting of two incubation steps was applied for Tg measurement. During the first incubation, standard Tg or Tg in serum was bound to the antibody coated cup. After washing, equal amounts of radioactive purified anti-Tg were incubated with the bound Tg. The cups were then washed again, and counted separately. The radioactive counts thus obtained, increased with the amounts of Tg bound to the anti-Tg by the first incubation.
The sensitivity of the assay was 4 ng/ml. T₃ and T₄ did not cross-react against Tg, and did not interfere with the binding between Tg and ¹²⁵I-anti-Tg. A dilution curve was constructed using the serum of a patient with Graves' disease ; the post-operative serum contained a very high level of Tg. This curve paralleled the standard curve. By adding constant Tg to a normal serum or Graves' serum, Tg recovery was good. However, in regard to Hashimoto's serum, Tg recovery was unsatisfactory.
4) Serum Tg concentrations were measured in patients with various thyroid disorders. Tg was detectable in 60% of 25 normal subjects tested, with an average level of 26.5±26.5 (mean±SD) ng/ml. Serum concentrations in 23 simple goiter, 9 untreated Graves' disease, 27 treated Graves' disease, 10 Hashimoto's thyroiditis having undetectable anti-Tg, 13 adenoma and 8 thyroid cancer were measured as 37.9±87.0ng/ml, 324.3±517.8 ng/ml, 71.0±138.6 ng/ml, 172.5±314.5 ng/ml, 126. 0±146.0 ng/ml and 15.0±14.1 ng/ml, respectively. In two patients with subacute thyroiditis, one patient had 100 ng/ml and the other gave a value of 280 ng/ml.
In patients with Graves' disease, there was no correlation between Tg concentration and T₃ and T₄. In two patients with Graves' disease undergoing partial thyroidectomy, serum Tg levels went up 16-30 μg/ml after the surgery. A patient with thyroid cancer showed a serum Tg value of 350 ng/ml after total thyroidectomy.
5) High Tg levels were observed mostly in patients with low titers of anti-Tg. However, even with the existence of potent anti-Tg, Tg could be detected in some instances. It seemed that these Tg values represented free Tg concentration when used in the present direct RIA system.

抄録全体を表示

The Leucocyte migration inhibition test (LMT) using the agarose plate technique according to Clausen was performed in 19 patients with Hashimoto's thyroiditis, 23 patients with Graves' disease, and in 17 normal subjects.
A series of leucocyte suspensions was preincubated at 37°C for 30 min. with crude thyroid extract (Crude), purified thyroglobulin (Tg) and thyroid microsomes (Micr) in concentrations of 500,300 and 100μg/ml, respectively, together with phosphate buffer saline (PBS) as the control. Then, each of the aliquots of 7μl of leucocyte suspension containing 1.5 million leucocytes, preincubated with various antigens or PBS, was placed into 4 wells, 2 in each of 2 agarose plates, followed by a 2 nd incubation for 24 hours at 37°C, in 2 per cent CO₂ with 98 per cent atomospheric air, resulting in a pH of between 7.2 and 7. 4. After the incubation, the migration areas were studied and measured by planimetry.
The degree of migration inhibition was expressed as a migration index (MI); that is, the ratio of the average extent of migration of the leucocytes cultured with antigens to that of control.
The MI values for normal subjects against Crude, Tg and Micr were 97. 6±6. 8 (mean±S. D.) %, 97.8±4.2%, and 95.5±5.3%, respectively. Low MI values, below 2 S. D. of the normal, were regarded as positive results.
The rate of positive LMT in patients with Hashimoto's thyroiditis were 32%, 46% and 31% against Crude, Tg and Micr, respectively. In Graves' disease, they were 35%, 50% and 41% for the same antigens, respectively. In 68-68% of patients with Hashimoto's thyroiditis or Graves' disease, positive LMT was at least shown with one of the three antigens.
In Graves' disease, there was no correlation between MI values and the presence of long acting thyroid stimulator (LATS). In untreated patients with Hashimoto's thyroiditis and Graves' disease with a short clinical course, the migration inhibition factor against Tg or Micr was found to be positive in a remarkably high rate. On the other hand, the levels of circulating antibodies in the serum tended to show high titers in patients with long duration, comparing with those with short duration.
From the histological findings obtained by needle biopsy of thyroid tissue, grade of lymphocytic infiltration was found to be significantly correlated with the degree of reduction in MI values.
From these observations, it is concluded that MIF against Tg can easily be detected by the agarose plate technique and that cellular immunity may play a more important role in the initial phase of autoimmune thyroid diseases than the later phase, in which the serum levels of circulating antibodies are becoming predominant.

抄録全体を表示

The copper-IUD has been proved to be more effective for contraception than the popular IUD. To understand this mechanism of action, the effects of metallic ions on estrogen and progesterone receptors were examined in the cytosol of estrogen primed white female rabbits. The 274,200×G supernatant of the uterine homogenate was used as the cytosol.
The cytosol and ³H-progesterone (3. 12×10^-9M) or ³H-estradiol-17β (3.46 x 10^-9M), with or without various metallic ions of different concentrations had been incubated for 2 hours. Bindings of steroids were estimated by the dextran coated charcoal assay (0. 001% dextran and 0.1% Norite A) and were evaluated by 5-20% sucrose linear gradient centrifugation.
1) Effect of various metallic ions on steroid hormone-receptor binding (determined by dextran coated charcoal assay. Fig. 1) : The steroid hormone-receptor interactions were markedly inhibited by Cu⁺⁺, Fe⁺⁺, and Zn⁺⁺ ions and moderately by Mn⁺⁺, but K⁺ and Ca⁺⁺ increased or slightly affected the binding at concentrations between 10^-2M and 10^-4M. There were some differences between estrogen and progesterone receptors in their sensitivities to various metallic ions.
2) The effect of copper ion on the binding of steroid to the receptor : The dextran coated charcoal assay (Fig. 2) demonstrated that the inhibitory effect of Cu⁺⁺ appeared at 10^-6M and the steroidhormone-receptor bound decreased down to 10% at 10^-2M Cu⁺⁺. The estrogen receptor was less affected by copper than the progesterone receptor.
It was demonstrated by 5-20% sucrose linear gradient centrifugation (Fig. 3) that estrogen and progesterone receptors, which both sedimented at 8 S, were changed to more sedimenting forms in the presence of 10^-4M Cu⁺⁺, and were dissociated to 6. 5 S form with moderate loss of steroid hormone binding affinity in 10^-2M Cu⁺⁺.
The kinetic study (Fig. 4), determined by dextran coated charcoal, showed that Cu⁺⁺ was a competitive inhibitor against steroid hormone receptor bindings with Ki≅2.7.×10^-5M to estrogen receptor (Kd≅1.4 × 10^-9M), and with Ki≅5.1 × 10^-6M to progesterone receptor (Kd≅8.1 × 10^-10M).
These results indicate the inhibiting factor of copper is the direct interference at the steroid binding site of the receptor, resulting in the increase in the effectiveness of the IUD.
While progesterone receptor is more affected by copper ion than estrogen receptor, it is suggested that estrogen receptor survives longer than progesterone receptor and thus the biological effect of copper seems to be somewhat estrogenic.

抄録全体を表示