Metyrapone and reduced metyrapone, the main metabolite of metyrapone, are known as 11-β-hydroxylase inhibitors.
We developed a method for the determination of metyrapone and reduced metyrapone so that we could investigate closely the hypothalamo-pituitary adrenal axis (H-P-A axis).Our method for the determination of metyrapone and reduced metyrapone in serum by means of gas chromatography-mass spectrometry (GC/MS) is as follows :
One hundred μl of serum was diluted with 0.5 ml of distilled water or serum.
(Dilution curve with either distilled water or serum showed linearity up to a range of 10⁴ times dilution.)
The serum samples, containing a fixed amount (200 ng-2μg/tube) of the internal standard (thonzylamine hydrochloride) were alkalinized with 0.1N NaOH at a pH above 7.8. These mixtures were extracted three times with 2 ml of ethyl acetate, which were moved to and combined in another tube. Then these extracts with ethyl acetate were shaken well together with 2 ml of 0.1N HCl for 15 minutes.
After we discarded the ethyl acetate layer, the residual HCl-layers were alkalinized again at a pH above 7.8 with 0.3N NaOH and were again extracted three times with ethyl acetate.
The ethyl acetate layers containing metyrapone, reduced metyrapone and the internal standard were evaporated to dryness with a stream of nitrogen in a 37°C water bath.
Following the above procedures, reduced metyrapone was converted to a trimethylsilyl (TMS) derivative of reduced metyrapone in the mixture (TMCS : TMSI : BSTFA = 3 : 1 : 3) for the determination of serum reduced metyrapone. This silylated reduced metyrapone at C-1 position improved tailing on the gas chromatogram.
The final samples, containing metyrapone, TMS derivatives of reduced metyrapone and the internal standard, were subsequently applied to GC/MS.
The conditions of the GC/MS procedure were as follows : The glass column was packed with 3% OV-17 chromosorb W (HP) 80-100 mesh, the injection port was set at 270°C, the column temperature was 220°C, the ionization voltage was 70eV, the accelerating voltage was 3KV. Electron impact ionization was used for the measurement.
The areas of mass fragmentgram, measured at m/z 106 for metyrapone, m/z 180 for TMS-derivative of reduced metyrapone and m/z 136 for the internal standard, were used for the calculation of the serum concentrations of metyrapone and reduced metyrapone.
The calibration curve showed good linearity (γ=0.999; P<0.001) from 20 pg to 500 pg.
The final recoveries of metyrapone and reduced metyrapone from serum were found to yield 90 ± 5% for metyrapone and 85 ± 6% for reduced metyrapone.
The coefficients of variation of accuracy and precision were less than 8.40% in the range of 20 pg to 500 pg.
The lower limits of sensitivity to metyrapone and reduced metyrapone in this assay system were 20 pg/one shot (S/N ? 20), respectively.
The serum metyrapone and reduced metyrapone concentrations were analyzed pharmacokinetically, according to the one-compartment open system model, especially with regard to age-specific differences.
The pharmacokinetic values after oral and rectal administration of metyrapone were statistically proved to show no significant differences either among the various age-groups or due to the routes of metyrapone administration in children. However, significant differences were observed with regard to the ratio of the fraction of the administered dose of metyrapone to the apparent distribution volume of metyrapone (F/V). Since the parmacokinetic values of serum metyrapone metabolism of various age-groups were not found statistically different, these pharmacokinetic values could all be included in the same control group.
The pharmacokinetic mean values of metyrapone metabolism in all groups were as follows : the apparent first-order absorption rate constant was 1.329 h^-1,

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This study was designed to investigate the development of the hypothalamo-pituitaryadrenocortical function in children.
In order to study age-differences, normal subjects were divided into three groups by age (A : 10 y/o -15 y/o, B : 5 y/o -9 y/o and C : 6 m/o -3 y/o).
The time course levels of plasma ACTH, serum 11-deoxycortisol, 11-deoxycorticosterone, cortisol, corticosterone, metyrapone and reduced metyrapone after a single oral or rectal administration of metyrapone were measured simultaneously by radioimmunoassay and gas chromatograph-mass spectrometry. These data were statistically analyzed.
The degree of inhibition of 11-β-hydroxylase activity was expressed by usings S/SF+ × 10² and DOC/DOC+B × 10² as the inhibitory indices in the glucocorticoid pathway and mineralocorticoid pathway.
After the administration of metyrapone, both maximum levels of plasma ACTH and the area under the curve (AUC) of plasma ACTH were higher and greater in the A group than in the other younger groups (by AIC). The response of serum 11-doxycortisol to the increase in plasma ACTH was greater according to age groups A>B>C (by AIC). In marked contrast, the response of serum deoxycorticosterone to the increase in plasma ACTH was greater according to age groups C>B>A (by AIC).
These findings suggest that the hypothalamo-pituitary-adrenocortical function differs with age and also that the development of the glucocorticoid pathway differs from that of the mineralocorticoid pathway in children.
The ratio of the increase of the S/S+F × 10² value to the increase of serum tota metyrapone (metyrapone plus reduced metyrapone) levels was greater according to age DOC groups A>B>C (P<0.05). In contrast, the ratio of increase of the DOC/DOC+B × 10² value to the increase of serum total metyrapone levels showed completely opposite results (C>B>A : P<0.05).
The discrepancies in these inhibitory patterns of 11-β-hydroxylase activity suggest the maturation with age and the heterogenity of the 11-β-hydroxylase between the glucocorticoid and mineralocorticoid pathways.
After discontinuation of more than 1 month of long-term prednisolone therapy, the response of serum 11-deoxycortisol to the metyrapone test did not completely return to normal responses in the control subjects.
This finding indicates that the recovery of the hypothalamo-pituitary-adrenocortical function may require more than 1 month under such conditions.
In patients with ACTH deficiency, plasma ACTH did not respond to the administration of metyrapone; however, serum 11-deoxycortisol and deoxycorticosterone levels showed a significant increase over the normal serum circadian range of 11-deoxycortisol and deoxycorticosterone (P<0.01).
These findings suggest not only a minute amount of pituitary ACTH secretion but also the autonomous function of the adrenal gland.
Furthermore, in ACTH deficient patients, the inhibitory patterns of S/S+F × 10² and DOC/DOC+B × 10² to the increase of serum total metyrapone were indicated to be the same as in normal children.
When we analyzed those cases showing a false negative response on the metyrapone test (i.e. cases whose maximum plasma ACTH increase was below 154 pg/ml : M + 3SD circadian ranges of plasma ACTH levels in the controls), the serum total metyrapone levels were found to be below 1.7 μg/ml in all; thus serum maximum levels of total metyrapone in a single metyrapone test must be checked so as to be above 1.7 μg/ml in order to evaluate normal pituitary function.
In the clinical application of a single metyrapone test (50 mg/kg, rectal administration), the demarcation points between the normal control group and the groups with abnormal ACTH secretion were 6.9 μg/di for 11-deoxycortisol and 7.2 ng/ml for deoxycorticosterone.
To summarize;

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Metergoline, a potent serotonin antagonist, has proved to be effective in the treatment of hyperprolactinemic anovulation. On the other hand, some investigators have reported that bromocriptine, a potent dopamine agonist, is effective in the treatment of normoprolactinemic anovulation as well as hyperprolactinemic anovulation. But there are very few reports as to whether metergoline is effective or not in the treatment of normoprolactinemic anovulation. This study was designed to investigate the ovulation-inducing effect and the underlying mechanism of metergoline in women with normoprolactinemic anovulation.
Metergoline was administered 8-12 mg daily for 8-2 weeks to 28 patients without galactorrhea (7 cases with anovulatory cycle, 17 cases with 1st grade amenorrhea defined by responding to progestin with bleeding, and 4 cases with 2nd grade amenorrhea defined by responding to estrogen and progestin with bleeding), whose serum prolactin levels were under 25 ng/ml. The serum levels of LH, FSH and prolactin, and also the pituitary responsiveness to 100 μg of LH-RH and 500 μg of TRH were examined before and during metergoline administration in 13 amenorrheic patients. In patients whose ovulation or withdrawal bleeding was induced and in patients whose menstruation was not induced, these stimulation tests during metergoline administration were performed at 5-7 days from the onset of menstruation and between 6 and 8 weeks from the beginning of treatment, respectively. In 6 other amenorrheic patients, the positive feedback effect of estrogen (Premarin 20 mg iv) on LH release was examined before and during treatment. In addition, a 12 mg dose of metergoline was administered daily for one menstrual cycle to 5 normal cycling women, and the Premarin stimulation tests were performed before and after metergoline treatment.
Ovulation was induced in 14 out of 24 patients except those with 2nd grade amenorrhea (58.3%), i.e. anovulatory cycle, 85.7% (6/7); 1st grade amenorrhea, 47.1% (8/17), and in 29 out of 49 menstrual cycles (59.2%). Of a total of 38 menstrual cycles in 14 ovulationinduced patients, 29 were ovulatory (76.3%), indicating a high rate of repeated success. No ovulation was observed in 2nd grade amenorrhea, but slight increases in basal levels of LH and/or FSH and obvious elevations of serum estradiol-17β levels were observed during metergoline treatment, followed by the progesterone withdrawal bleeding in 2 out of 4 patients. The mean basal levels of LH and FSH showed no significant change. The serum LH and FSH responses to LH-RH were improved in some administration patients who responded poorly before metergoline treatment. Serum prolactin levels and the prolactin response to TRH were significantly lowered during metergoline treatment.
The long-term administration of metergoline improved or restored the impaired positive feedback effect of estrogen on LH release in patients with anovulation and resulted in more prompt and further LH release by estrogen in normal cycling women.
With these results we conclude that metergoline is effective in the treatment of relatively mild ovulatory disturbances and suggest that the induction of ovulation by metergoline may be mainly ascribed to the improvement or restoration of the impaired positive feedback mechanism of estrogen and pituitary gonadotropin secretion. However, it is unclear whether or not lowering normal levels of prolactin, per se, participates directly or indirectly in the mechanism of the induction of ovulation. Further investigation is needed to elucidate why the positive feedback release of LH and pituitary gonadotropin secretion were improved or restored by the long-term administration of metergoline in women with normoprolactinemic anovulation.

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Histopathologic studies were carried out on the thyroid tissues extirpated from 118 patients with Graves' disease at Ito hospital principally in 1977. The primary purposes of this study were to obtain the whole spectrum of histologic features of the thyroid tissues in Graves' disease and further to gain an insight into the precise clinical pictures of individual patients before, at and, if possible, after the operation.
Numerical assessment as expressed in terms of percentage in area was undertaken for each of 17 histopathologic findings which were selected under a functio-morphologic viewpoint and in consideration of generally accepted pathlogic findings for Graves' disease or its related diseases. These findings included characteristics of follicles, height and folding of follicular epithelium, degenerative changes of epithelial cells etc..
For cluster analysis, the Ward's method was successfully performed using assessment values of these findings. The resulting dendrogram showed seven groups (I, II₁, II₂, II₃, II₄, III₁and III₂). This grouping may be considered multifaceted, because it was arrived at by analyzing values of a number of histologic parameters. These values were also analyzed by the principle factor analysis method, and five factors were extracted. The obtained contribution rate of the first factor was 23.5%, the second 16.8% and the third 10.1%. Besed on the factor loading values calculated for each of these factors, the first factor (F-1) was interpreted to represent “inflammation factor”, the second (F-2) “epithelium proliferation factor” and the third (F-3) “cell degeneration factor”.
Concerning these factors, the factor loading scores were calculated for 118 cases and plotted on the two-dimensional planes formed by F-1 and F-2 and also by F-1 and F-3 in an attempt to visualize their general distribution pattern with respect to the histopathologic picture. In general, cases of the 7 groups by the cluster analysis occupied their respective regions on these planes. To grasp the clinico-pathologic correlation, duration of clinical course, serum antithyroid antibodies, preoperatively administered antithyroid drugs, and thyroid function were respectively grouped into a couple of classes. Each case as mapped on the factor plane showed a tendency to assemble as a group according to the clinical picture.
Small follicles lined by hyperplastic epithelium were frequently encountered in the cases of short duration, i.e. within six months prior to the operation, while large follicles encircled by flattened epithelium with a large amount of colloid were predominant in the cases of long duration exceeding six months. It was surprising to note that large follicles containing much colloid were often observed in cases with high levels of serum T₃, T₄, whereas it is generally accepted that a typical histologic finding associated with hyperthyroidism is the preponderance of follicles lined by high columnar cells and with a scant amount of colloid. Some of these patients with high levels of serum T₃, T₄ had been administered iodine drugs before the operation. The possibility can not completely be ruled out that a part of this association is merely spurious due to the potential dual effect of iodine, one exerting on thyroid histology to make it quiescent, the other exerting on serum hormone levels to elevate them.
Postoperative hypothyroidism frequently occurred in patients extirpated of a thyroid gland which consisted of small follicles accompanied by infiltration with lymphocytes and plasmacytes and moreover by formation of lymph follicles. Many of these patients with inflammatory findings in the thyroid gland had antithyroid antibodies in their sera. These findings may support the idea of categorizing a new entity called Hashitoxicosis.
However,

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A simple method for the estimation of a urinary steroids profile with glass capillary gas chromatography is described. A 10ml portion of a urinary specimen was hydrolysed by Helix pomatia juice. Additional hydrolysis with ox-liver beta glucuronidase was necessary for the complete liberation of the urinary steroids from their glucuronides conjugates. Liberated free steroids were extracted with ethyl acetate, and the extract was washed and evaporated under reduced pressure.
Finally extracted steroids were derivatised as MO-TMS (methoxime-trimethylsilyl) ethers. For the full-silylation of the steroids, the Sakauchi-Hornig method was employed and good results were obtained. An OV-101-WCOT column (30m length) was connected to the “solventless injection system” (a type of glass falling needle injection) and temperatureprogrammed gas chromatography was performed. By this method, eight fractions of 17-KS, Pregnanediol, Pregnanetriol, Pregnanetriolone, 5-Pregnentriol, tetrahydro-metabolites of 11-Deoxy-Cortisol and of Cortisol, and hexahydrometabolites of Cortisol were separated and quantitatively determined. Data processing was performed by an on-line microcomputer.
The value in normal male and female controls was compatible with the results of glass capillary gas chromatography measured by Shackleton.
Urine specimens from children with adrenocortical cancer and 21-hydroxylase deficiency were analyzed. Characteristic metabolic profiles of each patient were easily demonstrated. This method seems suitable for the routine clinical elucidation of abnormal steroid metabolism.

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A solid phase radioimmunoassay for the detection of anti-human thyroglobulin antibodies using ¹²⁵I-protein A has been developed and its clinical use was evaluated in 20 patients with Graves' disease and 20 patients with Hashimoto's thyroiditis. After coating polystylene tubes with human thyroglobuline (10^-2mg/ml), 50μl of sera were incubated for 2 hours at 25°C followed by the addition of ¹²⁵I-protein A. After incubation, the tubes were washed extensively to remove free¹²⁵I-protein A, and radioactivities of the bound ¹²⁵I-protein A were counted.
The results of this solid phase radioimmunoassay were compared with those obtained by two other commercially available kits : Thyroid test and Thyroglobulin antibody kit (¹²⁵I-HTg method). Based on the results of the ¹²⁵I-HTg and ¹²⁵I-protein A method, obtained from the sera of 26 healthy subjects, normal range of P/N₁ of the ¹²⁵I.-HTg method (P : patient, N₁ : anti-HTg antibody free serum) and P/N₂ of the ¹²⁵I-protein A method (P : patient, N₂ : mean of healthy subjects) were obtained. P/N₁ higher than 0.92 and P/N₂higher than 1.64 were considered to be positive for anti-thyroglobulin antibodies.
In Graves' disease, 7 out of 20 patients (35%) were positive by thyroid test. Positivity increased up to 10 (50%) by the ¹²⁵I-HTg method, and 13 (65%) by the ¹²⁵I-protein A method. Among 20 patients with Hashimoto's thyroiditis, 13 (65%) were positive by thyroid test, 17 (85%) were positive by the ¹²⁵I-HTg method, and 20 (100%) were positive by the ¹²⁵I-protein A method. Thus, sensitivity for the detection of anti-thyroglobulin antibodies by solid phase radioimmunoassay using¹²⁵i-protein A was much higher than those obtained by commercially available kits. The whole procedure of this method is easy to operate as well.
In conclusion, this solid phase radioimmunoassay using¹²⁵I-protein A is useful for the detection of anti-thyroglobulin antibodies.

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The effects of irradiation on both the cytosol glucocorticoid receptor and concentrations of corticosterone and cyclic nucleotides in the rat liver were investigated. The liver concentrations of corticosterone and cyclic nucleotides were measured by radioimmunoassay before and after the irradiation of 1,000 rad/1 fraction. The glucocorticoid receptor in the liver cytosol was determined by the measurement of the cytosol binding to ³H-dexamethasone. The cytosol and nuclear corticosterone levels reached a peak 1 day after the irradiation of the rat liver and declined to the control levels after 2 days. The increase in corticosterone levels may be due to the direct stimulation of the right adrenal gland and/ or the stress induced by the irradiation. The binding capacity of the glucocorticoid receptor in rat liver cytosol decreased to the minimum 1 day after the irradiation, and the recovery occurred at 4 days. The Kd value of the glucocorticoid receptor remained unchanged from 1 hour until 4 days but was high at 4 and 7 days. The distinctly increased levels of cyclic GMP in the rat liver were found from 1 hour through 7 days after the irradiation, while cyclic AMP did not change.
The inversed relationship between the cytosol glucocorticoid receptor and corticosterone levels in cytosol and the nuclei indicates that the receptor-bound corticosterone in cytosol can be transferred to a nucleus and remain there in the presence of appropriate amounts of corticosterone in cytosol, after which the receptor is released from the nucleus into cytosol. The high Kd values observed 4-7 days after the irradiation may be either due to the direct effect of irradiation or to the replenishment of the receptor with a low affinity.

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