Despite the widespread use of many different estrogenic compounds in hormonal contraception and estrogen replacement, in the human a simple test for comparing the potencies of the compounds is lacking. Many of the estrogenic compounds are difficult to measure, and animal studies may not relate directly to those done on humans in terms of potency. Corticosteroid-binding globulin (CBG) in human serum has been known since 1958 to increase in response to estrogen administration, and the degree of its elevation has been shown to be closely dose-related and reasonably sensitive. Therefore, CBG represents a biological parameter that provides for the specific quantal analysis of estrogenicity in the human. In this paper a simple and reliable method for the determination of the cortisol-binding capacity of CBG (CBG-BC) in human serum is described.
The procedure is based on the amount of cortisol required to saturate the CBG binding sites. All endogenous steroids are initially removed from the serum by adsorption on dextran-coated charcoal (DCC). A mixture of a known amount of unlabeled and labeled cortisol is added to the sample in order to saturate the “stripped” binding sites of CBG. The unbound cortisol is removed by DCC at 4°C, and the supernatant represents the total cortisol-binding capacity of the serum sample. To distinguish between cortisol-binding to CBG and to albumin, “stripped” serum is heated for 30 min at 60°C, the temperature which does not influence cortisol binding by albumin but which does inactivate CBG. The samples are subsequently equilibrated with a mixture of labeled and unlabeled cortisol. The free cortisol is removed by DCC and the bound cortisol remaining in the supernatant is designated as the albumin-bound cortisol. Thus, CBG-BC is assayed by a double adsorption technique as differential binding to native and heat-inactivated serum. The cortisol deter mined by this method corresponds to CBG-bound cortisol, and CBG-BC is expressed as micrograms of cortisol bound per 100ml serum. The precision and accuracy of the method were found to be good.
The values of CBG-BC obtained by this method were 14.9±1.9 (SD) μg/100ml in adult men and 16.0±1.8 μg/100ml in adult women. Daily serum samples in five women revealed no significant variations in CBG-BC throughout the normal menstrual cycle despite fluctuating levels of serum estradiol. CBG-BC was also found not to vary significantly with age. In pregnancy, however CBG-BC increased significantly.

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A simple and practical radioimmunoassay (RIA) for pregnanediol without any pretreatment of urine specimens such as hydrolysis and/or extraction was developed.
Antisera were produced by immunizing rabbits with pregnanediol-3-glucuronide (P-diol-3-G) -BSA conjugates and P-diol-3-hemisuccinate-BSA conjugates. The specificities of the antisera were examined by their cross-reactivities with related steroids. Antiserum to P-diol- 3-G-BSA was significantly cross-reacted with 20a-OH-P (201%), P-diol (100%), P-diol-3-G (77.3%, where 119% as free P-diol), to a lesser extent with 200-0H-P (17.8%), and did not react with other steroids. Antiserum to P-diol-3-hemisuccinate-BSA, on the other hand, was capable of reacting with P-diol (100%), P-diol-3-G (46.9%), 20α-OH-P (75.2%) and 20β-OH-P (8.97%), respectively. Antiserum generated against P-diol-3-G-BSA was almost twice as high in its avidity for P-diol-3-G as compared with antiserum raised against P-diol-3-hemisuccinate-BSA. Antiserum to 20α-OH-P-3-carboxy-methyl oxime-BSA conjugate was only reactive with 20α-OH-P and not with P-diol or P-diol-3-G. From the data presented here, antiserum to P-diol-3-G-BSA with high specificity toward urinary P-diol-3-G was used for the present assays.
Ammonium sulfate precipitation was employed for the separation of antibody-bound and free tracer hormones. A direct method in which simply diluted urine was directly applied to RIA and the enzymatic hydrolysis method which requires extraction of P-diol with ether following hydrolysis of urine by β-glucuronidase were both examined in the present RIA on the same specimens which contained known amounts of ³H-P-diol-3-G.
Values obtained by the direct method were in good correlation with those determined by the hydrolysis method (r=0.994 p<0.01, Y=1.07X + 0.30). The working range of the assay was from 50pg to 5ng of P-diol per tube, and intra- and interassay precisions were 9.8, 12.5 and 12.9, 16.7%, respectively. Determination of radioimmunoassayable P-diol-3-G in urine was also in good correlation with the values obtained by the currently used analytical procedure, the Klopper-Kambegawa method.

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It has been reported that Xenopsin, Neurotensin and Substance P change plasma glucagon and insulin levels when they are administered in vivo. In order to clarify whether these agents have a direct effect on glucagon and insulin secretion from the pancreas, the action of each substance was examined by using the rat pancreas perfusion technique. The results were as follows : The perfusion with 1 and 5 nmole/min of Xenopsin for ten minutes resulted in a significant but transient release within two minutes. Neurotensin did not show any stimulatory effect on glucagon release in the concentration of 1 or 5 nmole/min for ten minutes. However, Substance P lowered significantly the glucagon concentration in the perfusate in a similar concentration. None of these substances influenced significantly insulin release from the perfused pancreas. These findings suggest that the hyperglucagonemia caused by these three agents in vivo may not be attributed to the direct effect on the pancreatic A-cell.

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It has been reported that TRH is able to secrete not only TSH but also prolactin. As a method to elucidate this relationship, the response of TSH and prolactin secretion to TRH was observed in puerpera in which the serum prolactin level was elevated, but serum TSH level remained at a normal level. Following TRH administration, serum prolactin and TSH levels were observed for 2 hours.
In puerpera, the serum prolactin level before TRH administration was 73.4 ± 52.0 ng/ml, and this value was extremely high as compared with 6.7 ± 4.9 ng/ml in normal female subjects. Following TRH administration, serum prolactin elevated to an extremely high level in puerpera as compared with that in normal female subjects.
In puerpera, the serum TSH level before TRH administration was less than 3.4 μU/ml, and this value was the same or less than 3.1 μU/ml in normal female subjects. However following TRH administration, in puerpera serum TSH elevated to a significantly high level as compared with that in normal female subjects. Namely, an increased TSH response to TRH was observed in puerpera with high prolactin levels.
These results suggest two hypotheses : firstly, there is an increased requirement for both prolactin and TSH secretion in puerpera, secondly, there is a latent hypothyroid state in puerpera.

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There is an increasing evidence that autoimmune mechanisms may have a role in the pathogenesis in insulin-dependent diabetics. The numerical and functional study of peripheral blood lymphocytes in diabetes mellitus might indirectly contribute to the understanding of its pathogenesis.
In this study, detection of peripheral blood T lymphocytes was measured by rosettes with sheep red blood cells (SRBC), and B lymphocytes were measured by immunofluorescence with specific antiserum to immunoglobulins.
The mean (± SD) percentage of SRBC was 67.6 ±7.2 in 21 normal subjects, 71.5±7.0 in 15 insulin-dependent diabetics, and 68.3 ± 6.7 in 30 insulin-independent diabetics. There was no difference in the absolute T-lymphocyte number per mm³ in these three groups. Insulin-dependent diabetics showed a normal percentage and absolute number of B lymphocytes when compared with normal subjects.

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A sensitive and specific double antibody radioimmunoassay for the measurement of serum thyroglobulin (Tg) has been developed. The minimum detectable concentration of Tg was 5.0 ng/ml. Coefficients of within and between assay variation were 2.4% and 12.0%, respectively. The mean recovery of Tg added to normal serum was 102.9%; and T₄, T₃, DIT and MIT did not crossreact in this assay system. Dilution curves of normal thyroid extract, tumor extract and patient's serum were shown to be parallel with the standard Tg preparation.
The mean serum Tg level in normal males and females was 42.8 ± 5.3 ng/ml (mean ± SE) (N=29) and 117.1 ± 20.9 ng/ml (N=20), respectively. There was a significant difference between male and female groups (p<0.001). The mean serum Tg level was 365.0 ± 69 ng/ml in 19 hyperthyroid patients with Graves' disease and 248.1 ±35.8 ng/ml in 21 patients who were in a euthyroid state from the treatment with antithyroid drugs, showing statistically no significant difference. However, 8 patients in permanent remission showed definitely low Tg values, 83.6 ± 16.2 ng/ml. The mean serum Tg level was 210.1 ± 57.6 ng/ml in 6 patients with chronic thyroiditis without auto-anti Tg and 525.1± 207.5 ng/ml in 5 patients with thyroid adenoma.
The effect of total thyroidectomy on the serum TSH and Tg was studied in a patient (M. T.) with pulmonary metastases from thyroid follicular adenocarcinoma. The serum TSH level rose progressively to hypothyroid levels during nine days after thyroidectomy; the value was 45.7 μU/ml on the 9th day after the thyroidectomy. The serum Tg level in this case was 4 925 ng/ml before surgery. After a transient fluctuation caused by the operation, the serum Tg level in the patient increased progressively during 3-9 days after surgery with a concomitant increase in serum TSH; the levels at the 3rd, 6th and 9th day after surgery were 5,825 ng/ml, 7,910 ng/ml and 11,190 ng/ml, respectively. The suppression of endogenous TSH secretion with treatment of T₃ 60 μg/day was followed by a gradual fall in serum Tg levels, decreasing to 630 ng/ml at the 114th day.
Bovine TSH was administered to this patient at the 114th day, so as to study the effect of exogenous TSH on serum Tg. Serum Tg reached a maximal peak at the 24 hr. after bovine TSH injection. The maximal increase of serum Tg above baseline was 221%. Despite complete removal of the thyroid gland, the increase in serum Tg after thyroidal stimulation with endogenous and exogenous TSH was observed in the patient. In addition, the increase in serum Tg after bovine TSH injection was also observed in two patients with differentiated thyroid carcinoma who underwent a total thyroidectomy and had only metastatic tissue. These results indicate that the elevated serum Tg was released from metastatic tissue by TSH.
The present study demonstrates direct evidence that metastatic tissue from thyroid carcinomas is responsive to TSH. The measurement of TSH responsive serum Tg in patients after thyroidectomy may lead to finding the presence of metastases from differentiated thyroid carcinomas. Furthermore, it seems possible to determine the effect of thyroid hormone therapy on the regrowth of metastatic tissue by the presence or absence of TSH-responsive increase of serum Tg.

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The present study was undertaken to investigate suppression of the pituitary-adrenocortical function by long-term corticosteroid therapy and spontaneous recovery from such a suppressant drug effect. Sixty patients who were on corticosteroids (hereinafter their dosage is expressed as the equivalent of prednisolone) given at 10 to 40 mg initially and then on a gradually decreasing basis down to below 10 mg currently or who had already been withdrawn from such a drug regimen were involved in the study and analysed for baseline values for plasma cortisol. These patients were evaluated for the functional status of the pituitary-adrenocortical system in relation to varying combinations of such factors as the total dose of corticosteroids, the duration in days of medication, the duration in days of medication at reduced dosage levels, and time in days elapsed from cessation of medication. The results led to the following conclusions :
(1) In patients receiving corticosteroids at such daily dosage levels as are reduced gradually to 7.5 mg or below within the duration in days as defined by the inequality Y≥8.4X + 222 (where Y stands for total dose given and X stands for the duration in days of administration), the pituitary-adrenocortical function is assumed to be in a state of being suppressed.
When corticosteroids have been given at daily dosages reduced gradually to 7.5 mg or below within the duration in days as determined by the inequality Y≤6.8X + 140 (where Y and X, respectively, stand as mentioned above), the pituitary-adrenocortical function of the recipient patient is considered to be in a state either of being not suppressed or of being recovered from suppressant drug effect.
(2) In cases where corticosteroid therapy is started with a moderate dosage, then reduced to a low dosage level or stopped within a relatively short period of time, the recipient patient can be safely withdrawn from the drug therapy without suppression of the pituitary-adrenocortical function if the dosage schedule is in line with the inequality Y≥5.9X + 331 (where X denotes the duration in days of medication and Y, total dose).
These results led the author to formulate a model of a corticosteroid dosage schedule which is reasonably free from the risk of causing pituitary-adrenocortical insufficiency. More particularly, the dosage of corticosteroids is reduced by a 5 mg decrement at regular intervals of 5, 8, 13, 27 and 103 days, respectively, when the initial daily dose is 30, 25, 20, 15 and 10 mg. By following this dosage regimen one might expect safe withdrawal from corticosteroid therapy without risking suppression of the pituitary-adrenocortical function.
(3) Corticosteroid therapy, when given at a dosage of 7.5 mg or less daily, is considered to have little suppressant effect on the pituitary-adrenocortical function. In cases where the pituitary-adrenocortical function has been suppressed as a result of corticosteroid therapy, the use of the drugs at a maintenance dose of 7.5 mg or less per day may be reasonably anticipated to result in spontaneous recovery of the suppressed function with avoidance of withdrawal syndromes.
(4) In cases in which the pituitary-adrenocortical function has been suppressed by the prolonged use of corticosteroids and daily dosage levels average less than 5 mg for the last one month, the same function is very likely to have already been recovered unless the total dose exceeds 1.6 g. Likewise, where an average daily dose of less than 5 mg has been given for the last 3 and 6-12 months, and if the total dose does not exceed 2.4 g and 4.9g, respectively, the pituitary-adrenocortical function in all probability has already been restored.

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An investigation was made using 97 clinically and chemically euthyroid relatives (T₃-RSU, serum T₃, T₄ and TSH; normal) in whom there were more than 2 thyrotoxic patients within the two degree relationship. The TRH test was performed in all 97 cases. In 56 of the 97 cases the T3 suppression test was also performed just after each TRH test.
The results revealed that 29 of the 97 cases (29.9%) showed an abnormal response to TRH. Fourteen of these (14.4%) revealed a negative or a hyporesponse (peak value of TSH was less than 4.9 μU/ml), and 15 cases (15.5%) revealed a hyperresponse (peak value of TSH was more than 35 μU/ml) to TRH. For the T₃ suppression test, 4 of 56 cases (7.1%) were nonsuppressible. Seven individuals who showed negative or hyporesponse to TRH consisted of two nonsuppressible subjects and five suppressible subjects. In 14 no or hyporesponsive cases, serum T₃ (1.51±0.05 ng/ml) and T₄ (9.91±0.31 μg/dl) were significantly higher compared with those of normal responders (1.30±0.04 ng/ml), (8.57±0.21 μg/dl) or hyperresponders (1.16±0.06 ng/ml), (7.77±0.63 big/dl). There was no definite correlation between negative or hyporesponders and nonsuppressible cases.
The following results were obtained :
1) In 29 of the 97 cases (29.9%), TRH tests showed abnormal response. Fourteen of these (14.4%) revealed a no or hyporesponse.
2) In the no or hyporesponsive cases, serum T₃ and T₄ were significantly higher compared with those of normal responders or hyperresponders.
3) Six of the 69 cases (8.7%) showed above 40% of the 24-hr thyroidal ¹³¹I-UPT.
4) For the T³ suppression test, 4 of the 56 cases (7.1%) were nonsuppressible, and in the nonsuppressible cases, serum T³ was significantly higher than that of suppressible cases.
5) We found a high occurrence of thyroglobulin and microsomal antibodies in our studies suggesting a linkage with hereditary predisposition.

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Several investigations in vivo and in vitro have shown that gastro-intestinal hormones stimulate insulin secretion. However, the reports on the insulinotropic activity of pancreozymin are contradictory. The conflicting results are probably due to the fact that pure native preparation of this hormone has not been obtained in “physiologic” doses. In the present study this problem has been investigated by exposing rat pancreas to caerulein in vitro. Caerulein, an active decapeptide isolated from the skin of the Australian amphibia Hyla caerulea, resembles pancreozymin in chemical structure, including C-terminus. This active polypeptide of nonmammalian origin has been shown to possess all the biological activities of pancreozymin. The present investigation was undertaken to evaluate the significance of the interactions of exocrine and endocrine pancreas using perfused rat pancreas in vitro.
Biphasic insulin release was demonstrated with caerulein at concentrations higher than 1 ng/ml. Insulin response of the first phase was proportional to the dose up to 1 μg/ml. The second phase of insulin release was, however, almost constant, regardless of the concentrations of caerulein. Release of glucagon was stimulated by the same concentrations of caerulein which stimulated insulin release.
Maximal responses of the pancreatic amylase and pancreatic juice output were observed with 1 ng/ml of caerulein. With higher doses, significantly less secretory responses were observed. The dissociation of the response to caerulein between endocrine and exocrine pancreas was found.

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