A simultaneous microdetermination of unconjugated estrone, estradiol, estriol and estetrol in serum or amniotic fluid by High Performance Liquid Chromatography with an Amperometric Detector is described.
Steroids in serum or amniotic fluid were extracted with 10 volumes of ethylether, and then ether extract was evaporated to dryness under N₂ gas. After defatting with a mixture of 50% methanol/n-hexane, the methanol phase was evaporated to dryness under N₂ gas.
The residue was applied to microcolumn packed with 2 ml volume of Sephadex LH-20 in the eluting soluvent benzene/methanol (85 : 15). Fractions contained estrone and estradiol; estriol and estetrol were collected and then evaporated to dryness under N₂ gas.
The sample solution was applied to HPLC using a reverse phase ODS column and acetonitrile : 0.1M KH₂PO₄ 47 : 53 for estrone and estradiol fraction, and 30 : 70 for estriol and estetrol fraction as a mobile phase, respectively.
The fraction of each estrogen was separated completely within a 20 minute period. The limit of detection of estrone, estradiol, estriol and estetrol was 50 pg, respectively.

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Simultaneous determinations of unconjugated estradiol (E₂), estriol (E₃) and 15α-hydroxyestriol (E₄) levels in maternal serum were studied serially to ascertain the significance of these estrogens in the feto-placental unit.
The samples of serum were collected serially from 25 normal and 44 abnormal pregnancies.
In normal pregnancy, these estrogen levels increased throughout pregnancy, especially E₃ and E₄ nearing the term. In 15 cases of IUGR pregnancy (including 4 cases of perinatal death), E₂ levels were mostly low (less than M-S.D.), E₃ was within normal limits (M±S.D.) or low, and E₄ was either high (greater than M + S.D.) or relatively low, and normal. In 9 cases of twin pregnancy, most E₂ levels were within normal limits, while E₃ and E₄ were remarkedly high.
The results signified that E₂ indicated placental function, that E₃ indicated placental and fetal function, and that E₄ indicated fetal function.

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A radioimmunoassay procedure for plasma 11-deoxycortisol (S) was developed using an antiserum prepared by immunizing rabbits with S-21-hemissuccinate bovine serum albumin and S-3-oxime-bovine serum albumin. Thereafter plasma S, cortisol (F) and adrenocorticotropic hormone (ACTH) responses to metyrapone were investigated in 13 normal adult males and 39 patients with prostatic cancer.
The results were as follows :
1) The antiserum against S-3-oxime-bovine serum albumin had less cross reactivity (<10%) with other steroids than that against S-21-hemissuccinate bovine serum albumin and obtained a good standard curve. The intraassay variance and interassay variance of this method using the former antiserum (N=10) were 12.4% asd 14.9% respectively, and the blank value was 3.7±1.6 pg.
2) Basal levels of S, F and ACTH in plasma from 13 normal adult males, ranged 21-80 years old, were 98.4±15.7 ng/dl (mean value±S.E.), 12.7 + 0.78μg/dl and 30.6±3.02 pg/ml respectively. Those levels increased to 7060±598 ng/dl, 24.3±1.69μg/dl and 24.3±1.6 pg/ml at 9 a.m. following oral administration of metyrapone (30 mg/kg b.w.) at midnight.
3) Both basal levels and responses of plasma S and F to metyrapone increased remarkably, while those of ACTH were within the normal range in prostatic cancer patients during the estrogen therapy. It was considered that protein-bound S and F increased following elevation of corticosteroid binding globulin but returned to the normal range about 2 weeks after discontinuation of the therapy.
4) In cases treated with estrogens, plasma S, F and ACTH responses to metyrapone were unchanged compared to normal adult males 2-4 weeks after discontinuation of the thereapy, and this data suggested that estrogens had no inhibitory effect on the pituitary-adrenal axis.
However, in cases treated with progestational agents over a long-term period, plasma S and ACTH responses to metyrapone decreased slightly but returned to the normal range 2-4 weeks after discontinuation of the therapy. This suggested that the inhibitory effect of these agents on the pituitary-adrenal axis was mild and reversible.

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Trilostane (WIN 24,540) blocks steroidogenesis by inhibiting the 3β-hydroxysteroid dehydrogenase system, and the effect has been produced in experimental animals and patients with certain kinds of hyperadrenocorticism. But the pharmacological profile of trilostane in the human adrenals has not been clarified, and the question exists whether or not this drug has an effect on the other steps in steroid biosynthesis. The present paper concerns the in vitro effects of trilostane on steroid production and conversion of tritiated pregnenolone to the other steroids in the adrenals.
Slices of adrenocortical tissue were used for the in vitro studies. Grossly normal adrenals were obtained from 32 cattles at slaughter and surgically from a patient with breast cancer. For hyperadrenocorticism, 2 adenomas and one hyperplasia were obtained surgically from 3 patients with Cushing's syndrome, one adenoma from primary aldosteronism and one adrenal carcinoma with hypermineralocorticoidism, respectively. Tissue slices weighing about 1.0 g were placed in 50 ml of a Krebs-Ringer bicarbonate buffer (KRB) containing glucose in a concentration of 200 mg per 100 ml. Tissues were preincubated for 1 hour at 37°C under a 5% CO₂-95% O₂ gas mixture shaking continuously at the rate of 150 cycle/ min. After preincubation, the slices were again incubated for one hour in 50 ml of fresh KRB-glucose to which approximately 1 μCi of 7-³H-pregnenolone (Specific activity; 10 Ci/ mmol) was added with or without trilostane at the concentration from 2×10^-8M to 1×10^-6M. Some of the human adrenal tissues were incubated without tritiated pregnenolone for the estimation of steroid concentrations produced during the incubation. After incubation, the media were decanted, extracted with dichloromethane and dried up in a vacuum drying oven under 60°C. The steroids converted from tritiated pregnenolone were separated by a thin layer chromatography developed in a medium of absolute toluene 90 : methanol 10 and radioactivity was detected by a thin layer chromatogram scanner. Then the areas of the strip corresponding to pregnenolone, progesterone, 17α-hydroxypregnenolone and 17α-hydroxyprogesterone, corticosterone, aldosterone and cortisol were respectively cut out and eluted with methanol for the tritium content count using a liquid scintillation counter. in vitro production of steroids without tritiated pregnenolone was analyzed by radioimmunoassay after an extraction and purification by column chromatography as reported previously.
The mean ratio of in vitro conversion of tritiated pregnenolone to other steroids was 65.3% (progesterone 25.7%, 17a-hydroxypregnenolone and 17α-hydroxyprogesterone 10.3%, corticosterone 22.0%, aldosterone and cortisol 4.1% and others 3.2%) of beef adrenals in the absence of trilostane. The conversion was reduced to 46.9% in the presence of 2×10^-8M of trilostane and to 18.9% at the concentration of 2×10^-7M of trilostane. Trilostane is an effective inhibitor of the 3β-hydroxysteroid dehydrogenase system in the beef adrenal gland. It is interesting that the doses of trilostane required to inhibit steroidogenesis in the present study were lower than those reported by Potts et al. (1978).
By adding 2×10^-6M of trilostane to the incubation medium for tissue slices from adrenal adenoma with Cushing's syndrome, the ratio of conversion from pregnenolone to other steroids was also reduced from 40% to 10.7% as beef adrenal slices. in vitro production of progesterone, 11-deoxycorticosterone, 17α-hydroxyprogesterone and cortisol by tissue slices from the normal adrenal cortex, aldosteronoma and adrenal carcinoma with hypermineralocorticoidism was markedly reduced by the addition of 2×10^-7M of trilostane.

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To investigate the pathogenic role of the sympathetic nervous system and the renin-angiotensin system in essential hypertension, we evaluated plasma catecholamine and PRA levels after glucagon stimulation and during one hour in upright posture after i.v. injection of frosemide.
In nine normal and high renin essential hypertensive (NHRH) subjects, i.v. injection of 1000big of glucagon caused rapid and significant increases in plasma epinephrine (E) concentration. Both the 5 min. level after glucagon injection (314±30pg/ml, mean±S.E., p<0.05) and peak value (358±87 pg/ml, p<0.05) were significantly higher than the basal level (170±25 pg/ml). Plasma norepinephrine (NE) concentration of the peak value (1065±231 pg/ml) was significantly higher than the basal level (357±50pg/ml, p<0.02). PRA levels increased in 4 out of 9 patients.
In seven patients with low renin essential hypertension (LRH), there was an impaired PRA response to glucagon, E also failed to increase at anytime after glucagon injection, but peak value of NE (1212±274pg/ml) was significantly higher than the basal level (391 ± 77 pg/ml, p<0.01). NE levels of NHRH during one hour in upright posture after i.v. injection of 40mg of frosemide were higher than LRH. Particularly at 10 min. level, the NE level of NHRH (895±115 pg/ml) was significantly higher than LRH (523±91 pg/ml, p<0.05).
These results suggest that the sympathetic-adrenomedullary function in LRH is diminished, and the renin secreting ability of JG cells is also diminished in LRH. The glucagon stimulation test is useful to assess renin secreting ability of JG cells.

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Enzyme immunoassay (EIA) for Thyroid Stimulating Hormone (TSH) has been developed. A principle of the assay was the competitive method and used a double antibody solid phase technique. TSH-horse radish peroxidase (HRP) conjugate and TSH-β-D-galactosidase (β-Gal) conjugate were used, and a fluorometry was introduced for measuring the activity of the enzyme. The substrates for determining the enzyme activities were 4-methylumbelliferyl-β-D-galactoside for β-Gal, and 3-p (hydroxyphenyl) -propionic acid for HRP. A polyacetal bead coated with a purified double antibody was used for the separation of Bound and Free.
With the assay methods, TSH in two of 3mm discs of dried blood on filter paper was measurable. The whole process was completed within 24 hours. TSH standards containing 0.05μU/tube showed 80-90% of B/Bo, and that containing 0.25μU/tube showed about 50% of B/Bo. As a routine method for mass screening of primary congenital hypothyroidism, the EIA method using TSH-β-Gal was adopted. A variation resulting from day to day experiments was 4.7-9.4% at B/Bo during a seven day trial.
From April of 1980, 12357 samples of newborn blood on filter paper were measured, and a distribution of the value of TSH in dried blood was almost the same as that obtained by RIA. Three cases of congenital hypothyroidism were detected, and about 0.15% of the total samples showed a TSH concentration higher than 20μU/ml of whole blood. All the samples were determined by RIA too, and the TSH values obtained from the EIA method were compared to that obtained from the RIA method. The correlation of both EIA and RIA methods was analyzed. Although there were slight discrepancies between EIA and RIA, the samples having high TSH values with the RIA method were all detected by the EIA method. Therefore, the EIA method will be usable as a tool for the mass-screening for the detection of primary congenital hypothyroidism.

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It is known that changes in the concentration of plasma catecholamine are closely related to the sympathetic nerve system and adrenal function. The determination of plasma catecholamine is, therefore, a very useful way to investigate the disorder of catecholamine synthesis and metabolism and also to diagnose certain diseases.
Catecholamine is a substance that exists in body fluids as a very small and labile component. Though several analytical methods of catecholamine are available at present and can be converted to the quantitative determination of plasma catecholamine, nothing has been admitted yet as an established determining method.
In this experiment we attempted to test whether or not a combination of high performance liquid chromatography with electrochemical detection could be used as an efficient method of plasma catecholamine determination.
The results obtained are as follows :
1) The effect of stabilizing reagents for the recovery of catecholamine : Five different reagents of a) ascorbic acid b) sodium thiosulfate c) EDTA-2Na d) sodium thiosulfate and e) sodium thiosulfate plus EDTA-2Na, were tested, and the reagent of sodium thiosulfate plus EDTA-2Na was concluded to be the most effective stabilizer among them. Moreover at this combination of the reagent, plasma CA was kept stable under - 20°C for 20 days.
2) The effect of protein precipitant : Perchloric acid was employed as a precipitant of the plasma protein. Among four different concentrations of 0.1, 0.5, 1.0 and 2.0N, the 0.5N concentration was disclosed to be the most effective concentration for the recovery of catecholamine in the supernatant.
3) The amount of alumina : Three different amounts of alumina (5, 10 and 50mg) were tested, but no difference was observed between 10 mg and 50mg. pH dependency of catecholamine adsorption on the alumina : Maximum adsorption was observed at pH 8.5 when the experiments were performed with 0.5M tris HC1 buffer.
4) The effects of voltage of electrochemical detector on the accuracy of the determination : Among six different voltages from 0.2 to 1.0 tested, 0.6V was concluded to be the most efficient voltage for the accuracy of determination.
5) DHBA as an internal standard of catecholamine : It was determined that DHBA could be used as an internal standard of the catecholamine.
6) The comparison between the double isotope method and this method : Results from the two different methods correlated quite well, indicating that the method reported in this paper can be used as a reliable quantitative determination of the plasma catecholamine, though the method seems to have several points that should be improved further.

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