It is generally accepted that plasma insulin levels (IRI) are low in patients with isolated ACTH deficiency. But, a comparison of IRI levels before and after replacement therapy has not yet been reported. We studied the insulin response to glucose and arginine administration (O-GTT, ATT) in 4 cases whom we encountered during the last 4 years. In O-GTT, 50g of glucose was given. In ATT, after 0.5g/kg arginine was infused intravenously, blood specimens were drawn at 0, 5, 15, 30, 60, 90,120 and 180 minutes. Plasma insulin was determined by a double antibody radioimmunoassay, and glucose (BS) was determined by an autoanalyser.
The results were as follows :
O-GTT : Before replacement therapy using glucocorticoid, in all 3 cases in whom the test was done, BS curves showed a normal pattern. Fasting plasma IRI levels and ∑IRI were low in 2 cases (cases 1 and 3). In the other one (case 4), plasma IRI levels at fasting, 120 and 180 minutes were undetectable. However, the peak IRI level at 60 minutes was very high (269uU/ml). One month after the initiation of replacement therapy ∑Bs increased in all cases. Fasting IRI, peak IRI levels and ∑IRI also increased in 2 cases (cases 1 and 3). In case 4, although fasting IRI increased, peak IRI level and ∑IRI markedly decreased.
ATT : Before replacement therapy, BS curves showed low responses in all 4 cases. Plasma IRI peaks were observed at 5 minutes in all cases, and they showed low or low normal. During therapy, BS peak levels and plasma IRI peak levels increased in all cases, especially in case 4.
From the above results, the insulin response to O-GTT and ATT in patients with isolated ACTH deficiency were divided into 2 groups. One showed concordance with the results in O-GTT and ATT before and after replacement therapy. The other showed dissociation from the results in O-GTT and NIT before and after replacement therapy. It was supposed that the differences in insulin response might be caused by a localization of the lesions in the brain.

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The present study was designated to examine the effect of adrenergic agonism on portal IRS levels in rats. Forty-five min after nembutal anesthesia, the rats were infused with 10μg epinephrine (EP), 0.5mg phentolamin (PH), 10μg EP plus 0.5mg PH, 0.3mg Methoxamine (MT), 0.5mg propranolol (PR), 10μg EP plus 0.5mg PR, or 2μg isoproterenol (IS) for 30 min. Two ml physiological saline was infused in a similar way as the control. Immediately before, and 15, 30 and 45 min after initiation of the infusion, portal blood samples were sequentially drawn from the same rats into the test tubes containing EDTA-Trasylol. The concentration of IRS was directly measured by a specific radioimmunoassay using ¹²⁵I- [Tyr⁸] -somatostatin as a tracer.
In the saline control group, the preinfusion levels of portal IRS were 249 ± 37pg/ml and slightly but significantly increased to 403 ± 50pg/ml at 30 min (p<0.05). The portal IRS levels at 30 min were increased to 1032 ± 164pg/ml by EP alone, to 1138 ± 240pg/ml by PH alone, and to 1682 ± 238pg/ml by IS alone. These values were significantly higher than each preinfusion level and the value of the saline control group at 30 min. When both EP and PH were administered together, the level by EP alone or by PH alone was raised further as high as 2136 ± 343pg/ml at 30 min. On the other hand, PR alone did not affect portal IRS levels, but PR administered concomitantly with EP completely suppressed the raised level by EP alone to 304 ± 31pg/ml at 30 min (p<0.01). When MT was given, the level of portal IRS at 30 min was 246 ± 32pg/ml. This value was significantly lower than that of the saline control group at 30 min (p<0.05).
These results show that the secretion of IRS to portal blood is stimulated by beta-adrenergic agonism and is inhibited by alpha-adrenergic agonism.

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One of the neurohypophyseal hormones, oxytocin, is known to have the biological activities of uterine contraction and milk ejection. Recently, serum levels of this hormone have been studied at several laboratories using the radioimmunoassay method, but reports dealing with the hypothalamic and pituitary regions where biosynthesis, storage and secretion take place, are rare. In this report, we will present the oxytocin content of the hypothalamus and hypophysis in Sprague-Dawley rats under various conditions, using a specific radioimmunoassay as previously reported.
In brief, an anti-oxytocin serum was made by immunizing New Zealand white rabbits with an oxytocin-bovine serum albumin complex, using Freunds' complete adjuvant. The antiserum was diluted to 1 : 9,000 for use in the radioimmunoassay. The chrolamine T method was used with some modifications for labelling ¹²⁵I to synthetic oxytocin, and the double antibody technique was used for separation of B/F hormones. In this assay system, the cross-reactivities of Arg-vasopressin and Lys-vasopressin were negligible, and the recovery rate was 102.5 ± 18.4% (mean ± S.D.). The values of the inter-assay coefficient of variation were less than 13.6%, and those of the intra-assay coefficient of variation were less than 7.5%.
Under ether anesthesia, the rats were decapitated, and the hypothalamus and hypophysis were removed. Isolated tissues were homogenized immediately with 2 ml of a 0.05 M phosphate buffer pH 7.5 containing 0.1M EDTA and orthophenanthroline under ice cold conditions. After overnight extraction at 4°C, the homogenate was centrifuged at 4°C for 20 minutes at 3,000 rpm. A sample of the supernatant was measured by radioimmunoassay, and the following results were obtained :
1) Dilution curves of the hypothalamic and pituitary extracts showed a parallel relation with the standard curve of synthetic oxytocin, and the same elution profiles were observed between these extracts and the synthetic oxytocin on a Sephadex G-25 column chromatography (1.2 X 90cm). From these results, the immunoreactive substance presented in the hypothalamic and pituitary extracts was considered to be the same molecule as synthetic oxytocin.
2) Oxytocin contents of the hypothalamus in male and female adult rats were 2.14 ± 0.59, 1.57 ± 0.32 mU/rat, respectively (mean ± S.D., n=10), and those of the hypophysis were 197.4 ± 30.7,169.2 ±18.2 mU/rat, respectively (mean ± S.D., n=10). There were no statistically significant differences between male and female rats in either tissue.
3) Pituitary oxytocin was measurable as early as the 4th day after birth; it then showed a marked increase with growth. Sexuality caused no difference at any age tested.
4) Hypothalamic oxytocin contents in pregnant rats showed a significant increase on the 3rd day of gestation (3.59 ± 0.79 mU/rat, n=10, p<0.001), as compared with nonpregnant levels, then gradually decreased towards the delivery date and reached minimum levels at the 20th day of gestation (1.29 ± 1.04 mU/rat, n=10). On the 4th day of postpartum, they increased again to the level of 2.75 ± 0.72 mU/rat (mean ± S.D., n=6), which was significantly higher than the nonpregnant levels (p<0.01).
5) Pituitary oxytocin contents in pregnant rats increased continuously from the 3rd day of gestation (377 ± 47.5 mU/rat, n=10) to the delivery date and reached maximum levels at the 20th day of gestation (492 ± 95.7 mU/rat, n=10). On the 4th day of postpartum, they remained at still higher levels (269 ± 62.9 mU/rat, n=6) than the nonpregnant levels.
6) Exogenously administered estradiol (50,100μg) or estradiol (100μg) with progesterone (25 mg) could not change the oxytocin content of either the hypothalamus or hypophysis in castrated or noncastrated rats.

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To examine the immunobiological implication of sex steroids during pregnancy, the effect of sex steroids on the incorporation of ³ H-thymidine into lymphocytes was investigated in both phytohemaggltinin (PHA) -induced blastic transformation of lymphocytes and the two-way mixed lymphocyte reaction (MLR).
Heparinized venous blood was taken from informed normal healthy men and nonpregnant women who were not taking oral contraceptives. Lymphocyte suspensions were prepared on a ficoll-metrizoate gradient centrifugation, and quadruplicate cultures were performed in flat-bottomed microtest plates. A culture mixture (0.2ml) containing 10⁵ responder lymphocytes, 3μg of PHA and various concentrations of steroid or 7.5 × 10⁴ responder lymphocytes from two individuals and various concentrations of steroid was placed in each well. Cultures were conducted in culture medium (RPMI-1640) supplemented with 20% of heat-inactivated autoserum and incubated in a humidified atmosphere of 5% CO₂-95% air in an incubator at 37°C, for 3 days for PHA or 6 days for MLR. Before termination of culture, 1.25μCi or 0.5μCi of ³ H-thymidine was added to each well and incubation was continued for 24hr for PHA or 18hr for MLR. Incorporation of ³ H-thymidine into the DNA of lymphocytes was determined by a liquid scintillation counter. Results were expressed as average counts per minute of quadruplicate cultures. Sex steroid hormones tested were estrone, estradiol-17β, estriol, testosterone, androstendione, dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone and 20α-dihydroprogesterone, in the concentration range between 10^-1ng/ml and 10⁴ng/ml. Each steroid was dissolved in ethanol and diluted with medium. A preliminary experiment to test the effect of ethanol confirmed that the ethanol concentrations present in cultures had no appreciable effect on the incorporation of ³ H-thymidine.
To test the cytotoxicity of sex steroids toward lymphocytes, cell viability was assessed by trypan blue dye exclusion under conditions identical to those used for MLR.
Although the suppressive effect of progesterone on MLR was less apparent than that on the lymphocyte blastogenic response to PHA, it was statistically significant at concentrations above 2 × 10³ ng/ml. The suppressive effect increased with increase of concentration and reached a maximum at 10⁴ ng/ml. The reported concentration of progesterone in the placental tissue is between 2 × 10³ and 6 × 10³ ng/g wet tissue throughout pregnancy, which agrees well with the effective concentration of progesterone described above.
Estradiol-17β had an inhibitory effect on MLR at 10⁴ ng/ml but no apparent effect on the lymphocyte blastogenic response to PHA at this concentration. The reason estradiol-17βsuppressed MLR but not the lymphocyte blastogenic response stimulated by PHA is unknown.
On the other hand, 20α-dihydroprogesterone had a remarkable suppressive effect on the lymphocyte blastogenic response to PHA but no effect on MLR. The reason for this discrepancy is also unknown.
The other 6 hormones tested had neither a suppressive nor enhansive effect on the lymphocyte blastogenic response to PHA or allogeneic cell stimulation.
These results, together with the fact that the highest concentration of progesterone in the human placenta occurs in the earliest period of pregnancy, suggest that the nonspecific immunosuppressive property of progesterone could be an important factor in the initial development of the biological relationship between mother and fetus.

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Intrauterine devices (IUD) have become popular in recent years. However, their mode of action is still poorly understood. Several studies have demonstrated an increase in prostaglandins in uterine horns bearing IUDs in rats, sheep and hamsters. As one hypothesis, increased synthesis of prostaglandins within the uterine cavity was proposed. In the present investigation, the changes of prostaglandin (PGE₁ and PGF_2α) content in rabbit endometrium bearing IUDs were studied by injecting hCG intravenously and measuring serum unconjugated estradiol and progesterone before and after ovulation.
As an intrauterine device, silk No. 7 suture was inserted transabdominally into a single uterine horn of mature female rabbits 1 to 2 months before examination (the IUD-bearing group). The rabbits of the control group did not receive any surgical treatment (the untreated group). Endometrial tissue and blood samples were obtained at designated times before and after hCG administration. All the compounds were measured by radioimmunoassay.
1) In the untreated group, the PGE₁ content in the endometrium was raised 12 hours (P<0.05) and 5 days (P<0.01) after the administration of i.v. 100 IU hCG, while no increase in endometrial PGF_2α was found throughout the 5 days after the hCG administration.
2) In the IUD-bearing group, PGE₁ content in the IUD inserted horn was higher than in the contralateral horn before (P<0.01), and 12, 48 and 72 hours (P<0.05, each) after the hCG administration. On the other hand, PGF_2α content in the IUD inserted horn was higher than in the contralateral horn before and 12 hours after the hCG administration (P<0.05, each) and then decreased to the levels of the contralateral horn or the horn of the untreated group.
3) The levels of endometrial PGE₁ of both groups changed in parallel with the levels of serum estradiol, while the levels of endometrial PGF_2α showed a negative correlation to the levels of serum progesterone.
4) There were no significant differences between the serum estradiol and progesterone levels in the IUD-bearing group and the untreated group before and after the hCG administration.
These results suggest that the increasing period of endometrial prostaglandins in the IUD-inserted horn give results similar to the most effective timing of prostaglandin administration for contraception.

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Testosterone (T) and estradiol (E₂) are bound to serum proteins, and it is believed that this binding affects the biological activity of these hormones. Testosterone-estradiol binding globulin (TeBG) is the primary β-globulin which binds both T and E₂ specifically.
In recent years, various assay methods for determining TeBG capacity (TeBGC) and %-bound T, E₂ to TeBG (%T-TeBG, %E₂-TeBG) have been developed but most of them too laborious and complex to apply in practice.
We have developed a rapid and simplified method to determine TeBGC and %T, E₂-TeBG by means of a modified ammonium sulfate precipitation method;
1) Assay method for TeBGC
(1) to a series of assay tubes, add ³ H-DHT and 5ng DHT (control; 500ng DHT)
(2) dry up under gentle N₂ gas stream at 40°C
(3) add 0.1 ml of undiluted serum (both male and female) or diluted serum (pregnant) to the appropriate tube, and allow to incubate at 37°C for 30 min.
(4) add 0.5 ml of ice-cold 60%-saturated ammonium sulfate
(5) keep in ice bath for 15 min.
(6) centrifuge at 1500 xg at 0°C for 30 min.
(7) count 0.2 ml supernatant and calculate
2) Assay method for %T, E₂-TeBG
(1) to a series of assay tubes, add ³ H-T or ³ H-E₂ (control; with 500 ng T or E₂) Steps (2), (3), (4), (5), (6) and (7) are processed as in the TeBGC method except using undiluted serum in every case.
The values obtained were 9.9 ± 3.4 (mean ± SD) for male (expressed as ng DHT/ml), 15.3 ± 3.9 for female and 87.1 ± 16.9 for pregnant. While %T-TeBG was 61.6 ± 5.1% for male, 76.8 ± 2.8% for female, and 92.4 ± 4.3% for pregnant, %E₂-TeBG was 25.0 ± 4.0% for male, 48.1 ± 3.4% for female and 80.3 ± 6.3% for pregnant.
The values thus obtained for %T-and %E₂-TeBG by the author's ammoniuum sulfate precipitation method are highly correlated with those which are determined by the equilibrium dialysis method. Correlation coefficients are 0.99 for %T-TeBG and 0.97 for %E₂-TeBG.

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Estradiol (E₂) binding capacity to plasma protein was estimated by simplified methods. Three techniques of dextran coated charcoal (D.C.), ammonium sulfate (A.S.) and concanavalin A sepharose (Con A.) were compared to separate bound and unbound hormones.
The D.C. and A.S. methods proceeded as follows: Purified ³H-E₂ in benzene-methanol was evaporated under nitrogen gas, and a half ml of sample plasma was added. Then, 0.4 ml of D.C. or A.S. was added to the plasma after one hour incubation at 37°C. Separation of protein bound E₂ was performed by centrifugation of 3000 r.p.m. at 4°C for 30 minutes. The supernate was counted by a liquid scintillation gamma counter.
The following procedure was performed in the Con A. method : Sample plasma of 0.1 ml and Con A. of 0.3 ml were mixed for one hour. Then, ³ H-E₂ was added and mixed for 30 minutes and settled for two hours. The supernate and/or sediment were counted. This procedure could be performed at room temperature.
Reproducible results were obtained when the prepared concentration of D.C. was 0.5%, A.S. was 50% and Con A. was 50%. The highest binding capacity was observed in alpha globulin fraction compared with beta and gamma globulin. The observed binding percents in alpha globulin were 33.5% by D.C., 78.1% by A.S. and 55.6% by the Con A. method.
A significant decrease of binding percent was observed in alpha globulin fraction when the protein solution was preincubated with cold E₂, suggesting the responsible role of alpha globulin in E₂ binding.
Plasma obtained from normal men, and women with normal periods, pregnancy, first degree amenorrhea and second degree amenorrhea were examined by the three methods. The D.C. method showed a notable difference between these plasma samples. The obtained percent bound was 84.6% in pregnancy of late trimester, 74.5% in normal women, 60% in amenorrheas and 45% in normal men.
These results suggest that alpha globulin might play an important role in E₂ binding, and the D.C. method could be suitable for the routine study of estradiol binding in women of different estradiol levels.

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SQ 14,225 (captopril), an orally active converting enzyme inhibitor, was administered alone or in combination with diuretics to 32 hypertensive patients with different etiologies for 1 to 12 months periods. Twenty-one of these patients had essential hypertension, 4 had renal failure, 2 suffered from renal parenchymal, 2 had malignant, and 1 suffered from renovascular hypertension. One had Cushing's syndrome, and one had a renin-secreting tumor. The daily dose of captopril was from 37.5 to 450 mg per day. The long term hypotensive effect and the correlations of the blood pressure changes and the pre-treatment renin dependency were evaluated. In order to investigate the involvement of factors other than the blockade of the renin-angiotensin axis in the hypotensive mechanism of captopril, endocrinological studies were done on 15 patients who had completed a one year treatment with captopril. The effect of indomethacin on the hypotensive action of captopril in these patients was also studied.
A sustained blood pressure reduction was achieved in most of the 15 patients for over a one year period of treatment, and no severe adverse reactions were observed. The blood pressure reduction significantly correlated with pre-treatment plasma renin activity (PRA) and blood pressure response to the infusion of angiotensin II antagonist within 1 and 2 months of administration, but was no longer noticed after 4 months of treatment. In addition, the correlation of blood pressure changes and the changes in plasma aldosterone concentrations (PAC) also gradually decreased (p<0.0025, 0.005, 0.05, n.s. at 1, 2, 4 and 12 months, respectively). The following measurements were compared among the groups of the 15 patients treated with captopril alone or in combination with diuretics, normotensive patients, hypertensive non-treated patients and hypertensives treated with thiazide alone for at least one year, with regards the renin-angiotensin system (PRA, PAC, plasma angiotensin I and II, urinary aldosterone excretion), serum converting enzyme activity, plasma and urinary catecholamine (adrenaline and noradrenaline), plasma bradykinin, and plasma prostaglandin E and F. Both captopril-treated groups had a significant elevation of angiotensin I and a reduction of aldosterone excretion when compared with the other groups. In spite of a sustained reduction of blood pressure, plasma catecholamine did not show a significant elevation in both captopril-treated groups, and urinary catecholamine was suppresed in the captopril-alone group. Serum converting enzyme activity, plasma bradykinin, kallikrein and prostaglandins did not differ among all groups. However, the administration of indomethacin (150mg/day for one week) significantly abolished the hypotensive action of captopril. From the present study, the sustained reduction of aldosterone excretion and the elevation of serum potassium indicate the persistent blockade of angiotensin II as one of the hypotensive factors in the long-term treatment with captopril. However, since the correlations of the changes in blood pressure and the pre-treatment renin dependency gradually diminished in spite of the significant fall in blood pressure, other hypotensive mechanisms should be considered. Although plasma prostaglandins did not show a significant elevation, the fact that indomethacin blunted the hypotensive action of captopril suggests that renal or local prostaglandins may play a role in the long-term efficacy of captopril. Sympathetic activity may also be suppressed by captopril and participate in its hypotensive efficacy.

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Primary aldosteronism is characterized by hypertension, signs and symptoms of potassium depletion, suppressed plasma renin activity and increased aldosterone production, and it is based on the presence of aldosterone-producing adrenal adenomas.
Although it is of paramount importance that the diagnosis of aldosterone-producing adenomas inclusive of its localization be established prior to surgery, these adrenal adenomas are usually so small that a preoperative demonstration of the tumor is rarely succeeded by radiographical methods, which ordinarily include retroperitoneal pneumography, percutaneous bilateral adrenal phlebography, and radioactive ¹³¹I-cholesterol adrenal scinti-scann.
Even when the concentrations of aldosterone in plasmas obtained via percutaneous catheterization from the right and left adrenal veins are compared with each other, this method often fails to establish an accurate diagnosis of the localization of aldosteronomas because the specimens obtained are not fully supplied by only the adrenal glands.
A new method is therefore needed to improve the accuracy of preoperative diagnosis in order to further delineate many ambiguous cases. The new method must be capable of localizing unilateral adenomas in most cases and at times, though rarely, bilateral adenomas.
A comparison of the calculated ratio of aldosterone concentrations with the cortisol concentrations in plasmas obtained via catheterizations of right and left adrenal veins is expected to confirm the presence and localization of aldosteronomas.
In our study, aldosterone-producing adenomas were correctly located prior to operation in all of 12 patients, i.e., in 11 unilateral aldosteronomas and in one bilateral aldosteronoma. This result stemmed from a comparison of the ratio of aldosterone concentration with that of cortisol in plasmas obtained by percutaneous catheterization of the right and left adrenal veins.
In this study, the ratio of aldosterone concentration (ng/dl) to cortisol concentration (μg/dl) measured on the venous effluent from adrenal glands containing adenomas was greater than 5.0 × 10^-3.
This procedure, measurements of, and calculation of the ratio of aldosterone to cortisol concentrations on specimens obtained via percutaneous bilateral adrenal vein catheterization is a definitive test for confirmation of aldosterone-producing adenoma in ambiguous cases.

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