Folia Endocrinologica Japonica

The Determination of Oxytocin Levels in Posterior Pituitary and Plasma of Rats by a Radioimmunoassay

Shigeru AONUMA, Yasuhiro KOHAMA

1972 Volume 47 Issue 12 Pages 1008-1017,1003
Published: March 20, 1972
Released on J-STAGE: September 24, 2012

DOIhttps://doi.org/10.1507/endocrine1927.47.12_1008

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The method of a radioimmunoassay for oxytocin was developed. Antibody was produced in rabbits by the repeated injections of oxytocin-bovine serum albumin conjugate emulsified with Freund complete adjuvant, and ¹³¹I-oxytocin was prepared by the modified chloramine T method and purified with two successive gel filtrations through the column of Sephadex G-15. The radioimmunoassay was carried out by incubation of a mixture of antibody, ¹³¹I-oxytocin and oxytocin sample for 2 days at 4°C, using saline-0.01M phosphate buffer (pH 7.4) containing 0.25% egg albumin as a diluent buffer, followed by separation of antibody-bound hormone (B) from free (F) by the dextran coated charcoal method (or ammonium sulfate precipitation method). The radioactivity of F (or B) and total reaction mixture was estimated, and the contents of oxytocin were calculated from the percentage of B. The sensitivity was 10μμg (4μU). Crude oxytocin extracted from the posterior pituitary of cow according to Kamm's method equally cross-reacted to synthetic oxytocin used as a standard in this assay, without any interferences from any substances except oxytocin that was included in this extract. But the cross reactivity of synthetic lys-vasopressin to oxytocin was only 0.5%, and bovine serum albumin employed in the process of antibody production did not show any influences on the assay of oxytocin.
This radioimmunoassay method was applied for the determination of oxytocin contents in biological samples of rats. Pituitary samples were prepared by extracting with 0.25% acetic acid and by washing with ether. Plasma samples were prepared by extracting with cold acetone and 0.25% acetic acid, and by washing with ether. The reproductibility of oxytocin from plasma by this extraction method was 63%. The normal levels of oxytocin were ca 1.4 μg/mg in the posterior pituitary and ca 40μμg/ml in plasma, but not detectable in the anterior pituitary of rats. The oxytocin levels decreased to ca 1.1 μg/mg in the posterior pituitary and to ca 30μμg/ml in plasma 2 weeks after male and female rats were castrated. By this assay method it was confirmed that the plasma oxytocin levels during a reproductive cycle of female rats increase just before and after parturition and 1 minuite after suckling. In the determination of rat biological samples a result of bioassay by a rat uterine muscle contractian method was about 10% higher than this radioimmunoassay.

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Hypothalamo-Pituitary Adrenocortical Function in Nephrotic Patients during Corticoid Treatment

Kenji MATSUOKA

1972 Volume 47 Issue 12 Pages 1018-1032,1004
Published: March 20, 1972
Released on J-STAGE: September 24, 2012

DOIhttps://doi.org/10.1507/endocrine1927.47.12_1018

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The inevitable and harmful suppression of hypothalamo-pituitary adrenocortical function results from the corticoid administration. In order to check the iatrogenic adrenocortical dysfunction and to prevent this side effect, the periodical ACTH-Z tests before, during and after treatment for the 13 nephrotic patients were performed.
The adrenocortical function was examined by daily urinary excretion of Porter-Silber chromogen, Zimmermann chromogen and 17-KGS. Functional reserve of hypothalamo-pituitary adrenocortical system was examined by response of intramuscular injection of 25 units of ACTH-Z and then by response to oral administration of 3 gm of SU-4885.
Both the daily urinary excretion of corticosteroids and the response to ACTH-Z in the nephrotic patients before corticoid treatment showed no significant difference from those in normal adults. Poor responses to SU-4885 were observed in four patients before treatment.
There was no correlation between the daily excretion of the response value to ACTH-Z and the results of renal function in the patients.
In the course of treatment with betamethasone, urinary corticosteroid promptly de-creased. However, the response to ACTH-Z was gradually depressed. The mean “adrenocortical response value” in terms of the increments in urinary Porter-Silber chromogen showed the following values : 20.1±10.9 mg before the corticoid treatment, 16.1±7.4 mg in 2 weeks after administration, 10.0±6.9 mg in a month, 7.9±5.9 mg in 2 months, 7.3±5.0 mg in 3 months, 4.6±4.9 mg in 4 months, 12.0±5.1 mg in a week after discontinuing betamethasone and 14.6±7.5 mg a month later. Similar changes in the adrenocortical response value from the increments in Zimmermann chromogen or 17-KGS in urine were observed. However, Porter-Silber chromogen was verified to be the most reliable measurement for tracing the response of adrenal cortex during the betamethasone treatment.
After the dosage of betamethasone tapered at 1.0 mg or less per day, the daily excretion of urinary corticosteroids and the response value to ACTH-Z were gradually elevated.
After discontinuation of corticoid therapy, the recovery of suppressed adrenocortical function was observed in all cases. A rebound phenomenon was observed within a week. The response value to ACTH-Z after discontinuing corticoid therapy also showed improvement but showed no significant difference from the level before treatment of corticoid or from normal range. The response to SU-4885 in only three cases were significantly reduced.
No inverse correlation was observed between the daily corticosteroids excretion, as well as the response value to ACTH-Z after treatment, and the sum of administered doses of betamethasone, or the duration of corticoid therapy. Throughout the course of treatment, there was no case who showed clinical manifestations or laboratory findings indicating adrenocortical insufficiency.
From these observations, it is concluded that the ACTH-Z test before, during and after treatment with betamethasone is a reliable test to evaluate the adrenocortical reserve. The withdrawal adrenocortical insufficiency will be safely avoided in so far as “adrenocortical response value” to ACTH-Z remains within the above-mentioned ranges.

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Metabolism of Synthetic Corticoids in Rat Liver

Osamu YAMASHITA

1972 Volume 47 Issue 12 Pages 1033-1045,1006
Published: March 20, 1972
Released on J-STAGE: September 24, 2012

DOIhttps://doi.org/10.1507/endocrine1927.47.12_1033

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The possibility that the enhancement of the anti-inflammatory activity of synthetic corticoids might be attributable to the less rapid metabolism than endogenous corticoid, cortisol, was widely supported by a number of investigators. But the comprehensive metabolism of the synthetic corticoids has yet not been reported. Therefore, this report presents a study on the metabolism of synthetic corticoids in the liver of Sprague-Dawley strain male rats. The corticoids studied were cortisol, prednisolone, 6α-methylprednisolone, triamcinolone, paramethasone, dexamethasone and betamethasone. Isotopic corticoids examined were ¹⁴C-cortisol, ³H-prednisolone, ¹⁴C-prednisone and ¹⁴C-triamcinolone acetonide. Each compound was incubated with rat liver homogenate, with or without NADPH₂, or liver slices. In other studies, each isotopic corticoid was injected into the portal vein of rat, and then the liver of the rats was removed at 0.5, 3, 6 and 9 min. respectively, after the injection. After that the removals were bloted on filter paper and homogenized in 50% acetone. Metabolites obtained from the extracts of the incubation medium and livers were chromatographed, purified and identified.
1) The relationship between anti-inflammatory activity of synthetic corticoids and their rates of recovery in rat liver in vitro and in vivo was investigated. A correlation was found between high anti-inflammatory activity and the resistance of these corticoids to the metabolic attack in the liver.
2) The metabolites of synthetic corticoids were analysed and found to be 20-dihydro metabolite and 11-dehydro metabolite of each original compound, prednisolone, prednisone, 6α-methylprednisolone and triamcinolone.
3) The fairly rapid conversion of prednisone to prednisolone as well as cortisone to cortisol was observed.
4) The experiments using a portal injection method revealed that cortisol was metabolized more predominantly to the 20-dihydro derivatives than to the tetrahydro derivatives in the liver. Synthetic corticoids were not metabolized in the rat liver to the tetrahydro derivatives both in vitro and in vivo.

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Effect of Thyrotropin-Releasing Factor (TRF) on Pituitary TSH Secretion in Man

Part I. With Special Reference to the Standardization for TRF test

Masahiro SAKODA, Makoto OTSUKI, Hiroyoshi FUKATSU, Shigeaki BABA, Naoh ...

1972 Volume 47 Issue 12 Pages 1046-1060,1007
Published: March 20, 1972
Released on J-STAGE: September 24, 2012

DOIhttps://doi.org/10.1507/endocrine1927.47.12_1046

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TSH releasing activity of synthetic thyrotropin-releasing factor (TRF), 1-pyroglutamyl-l-histidyl-l-proline amide, has been demonstrated both in vitro and in vivo experiments. It is valuable to use synthethic TRF for diagnosis and treatment of hypothalamic-pituitary disorders because of the specific action of TSH release induced by synthetic TRF from the anterior pituitary gland.
TRF was administered in one of four ways including, (i) a single “push” of 25-800 μg given over 30 seconds, (ii) 200 μg infusion in 100-300 ml of saline over a period of 30 to 120 minutes, (iii) subcutaneous injection of 100-200 μg or, (iv) a single oral dose of 1-10 mg. The intravenous administration of TRF produced the greatest response in the shortest time as compared with the other method of TRF administration. It is recomended to use the “single push” method as a routine TRF test because of the reliability of TRF absorption and the easiness of administration.
Six different doses between 25 and 800 μg of TRF were administered intravenously to 24 normal persons. A significant dose related increase of TSH was observed up to 400 μg of TRF. We recomended to use 50-400 μg of TRF for a routine test because these doses of TRF lead to a dose-related increase of radioimmunologically measurable TSH and the response dose not reach the maximum level.
We measured PBI, T₃ resin sponge uptake (T₃RSU), thyroxine and plasma TSH levels after TRF administration. There was a rise in serum PBI in 4 out of 10 subjects while T₃RSU remained unchanged after intravenous administration of TRF. Oral administration of 5 and 10 mg of TRF increased serum thyroxine levels. It can be said that plasma TSH was the most accurate indicator for intravenous single push administration of TRF.
Although intravenous single push method was employed as a screening test for TSH secretion, based upon the results mentioned above, the mode and dose of TRF adminstration should be studied further for a more precise method of pituitary TSH reserve test.

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[title in Japanese]

1972 Volume 47 Issue 12 Pages 1061-1092
Published: March 20, 1972
Released on J-STAGE: September 24, 2012

DOIhttps://doi.org/10.1507/endocrine1927.47.12_1061

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