The separation, identification and estimation of steroids by gas chromatographic methods rests upon the use of thin-film column packings prepared from highly thermostable liquid phases and inactivated supports ; microgram or sub-microgram samples are used with highly sensitive ionization detection devices. Separations are usually achieved by using the appropriate liquid phase, and derivatives may be prepared to aid in the qualitative or quantitative aspects of the separation. Non-selective liquid phases in current use include SE-30, F-60 and JXR ; these are all siloxane polymers. Selective phases in current use include the siloxane polymers QF-1, CNSi and PhSi, and the polyesters NGS, EGSS-Z, and CHDMS. Many types of useful derivatives are known.
The indentification and estimation of steroid hormones in materials of biological origin requires the development of an isolation procedure for a steroid fraction as well as a suitable gas chromatographic method. The best way to compare isolation methods is by gas chromatographic techniques.
The separation and estimation of urinary 17-ketosteroids may be carried out through use of trimethylsilyl ethers as derivatives. Pregnanediol may be estimated in the same way. Testosterone in urine may be determined either as the free steroid or as the propionyl derivative. DHIA in serum may be determined through use of an ion-exchange column procedure and a gas chromatographic measurement. Methods for the determination of THE and THF have been published, but these are not entirely satisfactory. Estrogens may be determined as the trimethylsilyl ethers or as acetyl derivatives. These methods may be used to study steroid metabolic pathways, responses to adrenal stimulation and alterations in steroid patterns produced by drugs.
Other types of hormones may also be studied by gas chromatographic techniques. For example, the catecholamines may be separated as acetyl derivatives with a JXRCHDMS column.
Examples of separations of steroids will be presented.

View full abstract

During the past few years we have established what appears to be the principal biosynthetic pathways to pregnenolone in various steroid-producing tissues. Cholesterol is an important precursor which may be successively hydroxylated at positions 20α and 22R or, alternatively, the order of hydroxylation may be 22R followed by 20α. Either pathway leads to 20α, 22R-dihydroxycholesterol which is converted to isocaproic aldehyde and pregnenolone by an enzyme designated as pregnenolone synthetase. The present communication deals specifically with these pathways and their control in rat testicular tissue.

View full abstract

The patients with primary and secondary amenorrhea were endocrinologically examined, using the determination of urinary estrogens, 17-KS, PBI, BBT and several other tests. In 17 amenorrheic women there were slight differences in the amounts of PBI and 17-KS, although the estrogen excretions were somewhat different : in the primary amenorrhea 10.5±2.7 μg/day (mean of five cases), the secondary amenorrhea 16.1±4.2 μg/day (mean of twelve cases), the follicular phase of normal cycle 23.8±4.6 μg/day (mean of seven cases) and the luteal phase of normal cycle 46.2±8.9 μg/day (mean of seven cases).
Five cases were selected in our clinic among the patients suffering from functional amenorrhea. Estrogen was administered as a single injection of 2 mg. of estradiol benzoate, and after two or three weeks, Pregnant Mare Serum Gonadotrophin (PMS) 7,000-22,000 IU and Human Chorionic Gonadotrophin (HCG) 6,000 IU-9,000 IU were administered to induce the ovulation.
The urinary excretion of estrogens was determined as serially as possible by Brown's method which was slightly improved in chromatography.
The maximal output values of the classical estrogens occurred on the third or the fourth day after the injection and the individual variation of estrogen excretion pattern was observed :
In two cases (case 1, 2) the ratio of estrone + estradiol/total estrogens was decreased in a few days after the administration of estradiol benzoate. From these results it is presumed that the conversion of estrogens in vivo from estradiol to estriol went on smoothly. In addition, in these cases the ovulation was induced by the administration of PMS and HCG and the urinary estrogens were remarkably increased, to such amounts as three or four fold more than the values in normal ovulatory phase. On the other hand, in one case (case 3) the ratio remained at a comparatively high level for several days after the administration of estrogen ; ovulation could not be induced and the urinary estrogens were in low levels. In this case the ovaries were atrophic and only primary follicles were observed histologically.
In two cases of secondary amenorrhea with hyperthyroidism (case 4, 5), the patterns of urinary estrogens were varied. That is, in one case (case 4) the estrogen metabolism was presumed to be normal, and in the other case (case 5), however, the estrogen metabolism was bad, and after 10 days' administration of Methylthiouracil the estrogen was again administered in the same way as above mentioned, and the estrogen metabolism changed from bad into normal.
Further studies on those problems are necessary, however, and it is also reasonable to consider that the estradiol tolerance test is one of the valuable methods in research on ovarian dysfunction.

View full abstract

A method for the gas-chromatographic estimation of urinary pregnanediol (5β-pregnane-3α, 20a-diol, P-diol), pregnanetriol (5β-pregnane-3α, 17α, 20α-triol, P-triol), pregnanetriolone (58-pregnane-3a, 17a, 20a-triol-11-one, P-triolone) and pregnanetetrol (58-pregnane-3a, Hp, 17β, 20α-tetrol, P-tetrol) is described.
A certain amount of 24-hour urine of a healthy person or a patient of congenital adrenal hyperplasis (CAH), was adjusted to pH 4.5 with acetic acid, and added with 300 units of beef liver 8-glucuronidase per 1 ml urine. After incubation at 37°C for 48 hours, it was extracted twice with chloroform, which was washed with 4% NaOH and distilled water, dried with anhydrous Na₂SO₄, then evaporated to dryness. The dried residue was chromatographed through alumina column and the each fraction containing P-diol and P-triol was brought to gas-chromatography.
Alumina column chromatography was performed with the following solvents : (1) 0.5% Ethanol in benzene, 50 ml prewash for P-diol, (3) 2% Ethanol in benzene, 20 ml P-diol fraction, (3) 2% Ethanol in benzene, 30 ml prewash for P-triol, (4) 6% Ethanol in benzene, 40 ml P-triol fraction, containing also P-triolone and P-tetrol.
The volumes of solvents suitable for alumina we used, were determined by a preliminary experiment with standard steroids.
P-diol and P-triol fractions were dried and the materials were made trimethylsilyl ether (TMSi) through the following procedure : dissolved in 0.2 ml pyridine, added with 0.1 ml hexamethyldisilazane and 0.1 ml trimethylchlorosilane, capped tightly, and shaken in ten minutes or left in 37°C for a few hours. The above solution was dried under nitrogen stream and the residue was dissolved in tetrahydrofuran. A portion of the tetrahydrofuran was served for gas-chromatographic analysis.
In gas-chromatography, a 3% XE-60 colum was used for P-diol fraction and a 1.5 % SE-30 column for P-triol fraction, installed in a Model GC-1B Shimazu Gas-chromatograph with hydrogen flame ionization detector (about detailed conditions, see Tables and Figures). Retention time of standard steroids relative to cholestane is shown in Table 1.
As the results (Table 2), neither P-triolone nor P-tetrol were detected in the urine of healthy persons. In cases of CAH a remarkable increase in P-triol was noticed, and P-triolone and P-tetrol were detected in good amounts. By an administration of metopirone to a case of CAH, a more increase of P-triol with the disappearance of P-triolone and P-tetrol was discovered.

View full abstract

In 1889, Takenaka first described prevalence of goiter in Esashi, the seashore district of Hokkaido. Further studies on pathological and endemic views have been reported by Takeda, Miyamoto, Shimpo and Inoue. According to these data, the prevalence of goiter in the Hidaka District, Rishiri-Rebun Islands and Shakotan Insula was 8.9%, 36.3% and 10.9%, respectively. The goiterous patients in these areas take large amounts of Kombu, a sort of seaweed which is rich in iodine, as a sidedish or as seasoning for the usual diet.
Since 1960, systematic investigations of this peculiar goiter have been carried out. In this paper, prevalence and geographical distribution, the nutritional environment, intake and output of iodine, and therapy are reported as the first part of our study. 1. Prevalence and geographical distribution
Seven thousand nine hundred and seventy schoolchildren, 7 to 18 years of age, including 4,726 in the Hidaka District, 2,298 in Rishiri Isl. and 946 in Rebun Isl., were investigated. Size of goiter was classified according to Shichijo's criteria, Dieterle's modification.
The incidence of goiter was calculated 6.6%, 8.9% and 2.7% in Hidaka District, Rishiri Isl. and Rebun Isl., respectively. In each area, girls were predominant : the ratio of female to male was from 2 : 1 to 10 : 1. Control studies were performed on school children of Sapporo city, and the incidence of goiter was 1.3%.
The 2nd degree size of goiter was found in 75.2% among 539 goitrous patients, 3rd degree in 21.5% and over the 4th degree in 3.3%, but in the Hidaka District the enormous goiter, over the 4th degree, was found in 16 cases.
The incidence of the goitrous patients with family history of goiter was 4 to 5%.
The incidence of goiter reached the peak at 12 to 14 years of age in both sexes.
The thyroid gland was diffuse on palpation in 97.4% of 539 cases, and nodular in only 2.6%.
2. Clinical findings
All of the 539 patients revealed clinically euthyroid states : hyperthyroidism or hypothyroidism, hoarsness and dyspnea ; congenital deafness or mutism was not found.
3. Histological picture
Histological picture of the thyroid gland of 7 goitrous patients in Hidaka District was examined. The findings of Struma colloides macrofollicularis, that represent a enlargement of follicles and colloid storage without abnormal cell-infiltration, were seen in 6 cases, and Struma colloides microfollicularis or Struma foetalis in the other case.
4. Nutrition Survey
Intake of foodstuffs was investigated in 8 families whose occupation was fishing, chiefly seaweed “Kombu” collecting. Five families had a history of goiter and 3 had not. Daily intake of nutriments throughout the four seasons were calculated according to the method of National Nutrition Survey of Welfare Ministry. It was found that most nutritional materials such as protein, carbonhydrate and Vitamin-A, were sufficient. The seaweed, chiefly Kombu, of 16.1 gm. which amounts to three times the average of Hokkaido Nutritional Survey in 1961 was taken. Excessive intake of other foodstuffs containing a goitrogen were not recognized.
5. Urinary inorganic iodine excretion
Five cases of goitrous patients in Hidaka District were examined for urinary inorganic iodine excretion, that was measured by Gross's method, under the usual diet in this area. These patients excreted up to 23mg. of inorganic iodine per day, whearas control non-goitrous patients on a normal diet at Hokkaido University Hospital excreted 1.5mg. and those on a iodide-restricted diet excreted 150, μmg.
6. Response to treatment
Fifty cases of goitrous patients in the Hidaka District were administrated desiccated thyroid or L-Triiodothyronine. In 72.5%, a disapperance or decrease in size of goiter was obtained. A more effective response was noted in the 10-to 15-year old or those having a smaller size of goiter.

View full abstract

Recently, the influences of the hormones from hypophysis-thyroid system on glucose metabolism has been studied. On the other hand, in the clinical observation, it has been frequently observed that diabetes mellitus or a simple symptom of urinary sugar is accompanied with hyperthyrodism. However, morphological influence of the hormones of this system on the pancreatic islets is not well known. In order to clarify the influence of the hormones of this system on the islets and to investigate the relationships between the histological findings and their functions, the author carried out the following series of experiments.
Materials and Methods
In this series of experiments, rats were used mainly, and sometimes rabbits were used.
The materials were colected under the conditions as described below.
These animals were divided into five groups as follows :
a) Thyradin (Powder of dried thyroid) administered group : Dosages were 20 mg/100g/day for rats and 200 mg/head/day for rabbits.
b) TSH administered group : Dosage for rats was 10 JSU/100g/day.
c) Growth hormone administered group : Dosage was 10 mg/head/day for rabbits.
d) Thyroidectomized group.
e) Hypophysectomized group.
In order to carry out the morphological studies and planimetric measurements of pancreatic islets on the above mentioned animals, the following methods were used :
a) Gomori's Chromealum-Hematoxylin-Phloxin and Aldehyde-Fuchsin dying-method, b) dying of zinc, c) dying of SS-SH groups, d) measurements of the area of Langerhans' islets, e) beta/alpha cell ratio, and f) nuclear volumes of alpha and beta cells.
Results a) Thyradin administered group :
After the administration of 200 mg each day for seven days, slight decrease in the quantity of beta granula, margination of beta granula, decrease in the amount of zinc, and enlargement of nuclei were observed in Langerhans' islets. These results seemed to show that the release of insulin still exceeded its production, although the production was accelerated much more than that in normal state. Alph cells were not changed, apparently.
After the administration for thirty days, degenerative change of beta cells, remark-able decrease of beta granula, condensation of nuclei, decrease in the amount of zinc, and decrease in the amount of SS-SH groups were found. These findings support the suggestion that there is an exhaustion of beta cells after the course of hyperfunction.
For alpha cells, decrease of beta/alpha ratio and slight enlargement of the cells were observed.
In the case of rabbits which were administered thyradin for fifty two fifty six days, there was no difference in the findings from those in rats.
The hormones of the thyroid seemed to accelerate the function of Langerhans' islets and decelerate it later.
b) TSH administered group :
After the administration of TSH for seven days, decrease in the quantity of beta granula, decrease in the amount of zinc, decrease in the amount of SS-SH groups were observed. These results seemed to show the acceleration of the release of insulin. No enlargement of nuclei and nucleoli would show the absence of the acceleration in the production of insulin.
After the administration of TSH for thirty days, decrease in the quantity of beta granula and remarkable decrease in the amount of zinc seemed to show the acceleration of release of insulin. And also increase and decrease of SS-SH groups seemed to show the acceleration of biological production.
This acceleration of the function of beta cells seems to be a secondary effect of the influence of TSH on glucose metabolism through thyroid.
c) Growth hormone administered group :
After the administration of growth hormone of 10 mg/day for fifty two-fifty six days, decrease in the quantity of granula and zinc of beta cells, and the tendency to increase in the dimensions of Langerhans' islets were found. There was no change of nucleoli.

View full abstract

The changes of the adrenal cortex caused by corticosteroids and ACTH, were studied histochemically using T.T.C. (2-3-5 triphenyl tetrazolium chloride), one of the tetrazolium compounds. T.T.C. is a water soluble and colorless crystalline substance, changes unsoluble in water, soluble in oil, and forms a pink colored precipitation (formazan) by a reducing process. Zweifach et al. (1951) incubated the adrenal slices of rats in T.T.C. solution to produce formazan in these slices, and then made frozen sections. From their results, they reported that the findings of the T.T.C. reaction clearly showed the adrenal function. In Japan, the T.T.C. reaction was studied by F. Akasu, H. Fukushima etc.. H. Fukushima reported that the T.T.C. reaction was a specific stain for corticoids after ascorbic acid, adrenalin, SS group and SH group were excluded.
In our experiments, when an equal volume mixture of 2% T.T.C. and 1% sodium hydroxide was dropped into adrenalin chloride, ascorbic acid solution or hydrocortisone suspension, deep red sediment appeared immediately in these solutions. In the mixture of hydrocortisone acetate and the T.T.C. solution, the red color began to appear 5 minutes after the mixing, and the deep red sediment appeared in 20 minutes. No red sediment appeared in the mixture of dexamethasone phosphate and the T.T.C. alkaline solution, but the sediment appeared easily after heating the mixture. From these results, it was confirmed that T.T.C. reduced and changed red sediments with reducing substances in the alkaline range. It is suggested that in corticosteroids, an OH group attached to the 21-carbon atom has a reducing action (Tab. 1).
Animal experiments. Male Wister rats of 100-200gr. in body weight, were used. After various dosages of corticosteroids were administrated, these rats were killed by cutting their heads. The autopsy was done immediately, and both adrenals were removed. After the adrenals were weighed, the adrenals were fixed in neutral formalin solution, then T.T.C. reaction was peformed. While T.T.C. reactions of the normal adrenal of rats were clearly observed in the zona glomerulosa as beautiful pink colored fine granules, the reactions were most conspicuously present in the outer layer of zona fasciculata. But the reactions decreased gradually in the inner layer of zona fasciculata, and some gross granules were present near the border between the adrenal cortex and the medulla (Fig. 1).
In the adrenal cortex of rats given dexamethasone, the initial change was the flecked finding where the pink color was deep in a section and was light in other sections of the zona fasciculata. Then the reaction of the zona fasciculata decreased markedly, and finally became negative following the atrophy of the adrenal cortex. In the above adrenal cortex, the pink colored gross granules of the zona reticularis had a tendency to increase in size and in number to a certain extent. Also, the zona glomerulosa of the rats given a large dosage of dexamethasone for a long duration, frequently a had conspicuous and strange T.T.C. reaction while the atrophied zona fasciculata had a slight or negative T.T.C. reaction (Tab. 5, Fig. 2, 3). Both the zona glomerulosa and zona fasciculata of rats given cortisone, had a slight or negative reaction.
The zona fasciculata of the rats given ACTH, initially showed a decrease of the T.T.C. reaction, but later had a tendency to decrease to a sertain extent. In the process, it was observed that some gross granules of the zona reticularis initially decrease the T.T.C. reaction, but later had a tendency to increase the reaction to a certain extent. In the process, it was observed that some gross granules of the zona reticularis initially decreased, and finally disappeared.
From these results, it is considered that the T.T.C. reaction is a beneficial method to study the function and condition of the adrenal cortex.

View full abstract

Influence of the neurohypophysial hormones on the thyroid activities was investigated in thyroxine-treated rabbits and mice, measuring plasma and blood ¹³¹I concentration as their parameters.
(1) In the raabits, 2-hr intravenous infusion of Pitressin at the rate of 10-30 mU/min., increased the plasma PBI¹³¹ concentration or at least blocked its fall which continuously progressed during the preinfusion period in all animals investigated (12 animals).
Synthetic lysine-vasopressin induced the same effect in 2 animals infused at a rate of 30 mU/min and in 2 out of 5 rabbits infused at 20 mU/min.
The potency of Pitressin, therefore, may be higher than that of synthetic lysine-vasopressin. Plasma water content, indicated by hematocrit, was not influenced by vasopressin infusion.
No increase in plasma TSH levels, measured by McKenzie's Method (Yamazaki's modification), was detected during Pitressin infusion, though it caused a significant rise in plasma PB¹³¹I, suggesting the direct stimulatory action of vasopressin on the thyroid.
(2) In the mice, both Pitressin and synthetic lysine-vasopressin in doses ranging from 10mU and 20mU to 100mU per mouse, respectively, significantly increased blood ¹³¹I concentration at a 2-hr interval, approximately lineally in proportion to the doses. The response to Pitressin was greater than that to lysine-vasopressin. The stimulation was also observed in the hypophysectomized mice, indicating the direct action of the hormone on the thyroid.
Smaller doses of Pitressin, less than 2mU, and lysine-vasopressin, less than 4mU, depressed the blood ¹³¹I levels as compared with those after saline injection.
The suppressive effect was also seen in the hypophysectomized mice, though the extent was less than that in intact ones.
Oxytocin showed no effect on the blood ¹³¹I in the thyroxine-treated mice.
Thioglycollate destroyed both stimulatory and suppressive effects of lysine-vasopressin, but only the inhibitory one of Pitressin, which was also seen in the hypophysectomized mice. This indicates that this suppression is due to vasopressin's inherent effect and that Pitressin contains another active contaminant.
Exogenous vasopressin did not modify the decay curve of the blood radioactivity after the administration of¹³¹I-labeled-L-thyroxine, showing no influence of vasopressin on distribution space of L-thyroxine or its peripheral metabolism in mice under the experimental condition employed.

View full abstract