The organ culture technique seems to be one of the most helpful in vitro methods especially in endocrinological studies, because, when compared with the incubation method, it makes a fairly long term observations possible with minimal losses of native characters of original tissue under the simple experimental conditions.
The present experiments were designed to study the mechanism of thyrotropine (TSH) secretion and release from anterior pituitatries of rats in organ culture.
The anterior pituitaries of male Wistar rat (150 gm. body weight) were used. Each pituitary was hemisected with a blade and each half was cut into 4 explants. One half of the explants was used for the experiment and the other half was used as the control (“paired culture”). The culture was performed by Trowell's organ culture method with some modifications. The author used an ordinary desiccator designed for a simple incubator instead of Trowell's culture chamber. Each culture dish contained the equivalent of two anterior pituitaries from 4 (or 8) donor rats. The explants were placed upon a defatted lens paper supported on a tantalum mesh raft, and the dishes were placed in the desiccator at 37°C under continuous flow of 95%O₂-5%CO₂ and cultivated for 3 to 10 days. Each culture dish contained 6 c.c. of medium “199” (Difco) which included 100 I.U./c.c. penicillin and 100 μg/c.c. streptomycin. In some experiments, various concentrations of sodium-L-thyroxine (T₄) and/or the extract of rat or bovine hypothalamus as TRF (thyrotropin releasing factor) preparation were added to the medium.
The media were collected to be pooled separately and kept frozen at 20°C until assayed for TSH by the McKenzie method. At the end of each culture period, one of the explants was fixed in Bouin's fluid and stained with hematoxylin and eosin for the histological examination.
The most favorable condition for culture was determined and it was decided that each dish contained the equivalent of two anterior pituitaries in 6 c.c. of medium which was exchanged daily.
In such a condition, when pituitaries from intact rats were cultured, the high TSH level was maintained for 10 days in cultured media. After whole culture period, about two times as much TSH was present in the explants and media than that present in fresh pituitary tissue.
It was seen that thyroidectomy 14 days prior to the beginning of culture showed stimulatory effect, and subcutaneous injection of triiodothyronine (T₃) for 11 days gave the inhibitory action to the TSH level of cultured media. These observations show that the influence given to pituitary gland in vivo may be transferred into the pattern of pituitary reaction in vitro.
When TRF (bovine hypothalamic extract : Fraction D) was added to the medium from the third day of culture, TSH level in the medium rose in the early stage of culture, then that in the tissue rose in the later period. TRF originated from rat hypothalamus also exerted its effect in this organ culture system.
The inhibitory effect of T₄ in the media could be seen, and the higher the concentration, the more evident was the effect. This shows that the inhibitory action of T₄ has a doseresponse relationship. But when the donor rat was thyroidectomized two weeks before culture, its inhibitory effect could be observed only in its high concentration.
Then when TRF and T₄ were added simultaneously to the culture medium, the effect of TRF could not be detected throughout the culture period. This phenomenon seems to show that the addition of T₄ to the medium inhibited the response to coexisting TRF.

View full abstract

To confirm that the organic iodine compounds were not present in saliva or urine a tracer dose of I¹³¹-T₄ was injected intravenously into patients with various thyroid diseases. The saliva and urine were collected for one hour. Iodine compounds of saliva and urine were extracted with acid-butanol and concentrated using nitrogen. Ascending paperchromatography was applied on the concentrated samples using butanol, acetic acid and water (78 : 5 : 17) as developer. The paper strips were scanned using actigraph, then autoradiograms were performed, and then stained with ceric arsenite reagents. Ascending thin layer chromatography was performed using the same samples and developer as paperchromatography. Then the absorbents were stained with ceric arsenic reagents, were scanned using scintillation counter, and the percentage of radioactivity of each iodine compound was calculated. Columnchromatography utilizing the anion exchange resin Dowex 1 was performed using adjusted acetate buffer to various pH 3.6, 2.2, 1.4, by 0.2 M acetic acid and 0.2 M sodium acetate and 3N-NaBr. Then these samples were scanned. Chemical analysis performed by butanol and Blau' Reasent.
From these results, the presence of a significant amount of organic iodine compounds in urine was confirmed, but a tracer dose of organic iodine compounds in salive was detected.
From these findings, we used the salive to investigate the thyroxine deiodination. There was a high salivary percent-radioactivity in nontreated hyperthyroid patients and a low one in nontreated hypothyroid patients. But these values were normalized with therapy. The s/p ratio (ratio of salivary percent radioactivity in 60 minutes to palsma percent radioactivity at 60 minute) was as follows :
Euthyroid 0.121±0.034
Nontreated hyperthyroid 0.179±0.040 (p<0.01)
Treated hyperthyroid 0.097±0.026
Nontreated hypothyroid 0.070±0.015 (p<0.01)
Treated hypothyroid 0.101±0.011
Simple goiter 0.110±0.027
Next, the early phase of disappearance of intravenous injected I¹³¹-T₄ from the circulation (acute thyroxine disappearance rate or acute thyroxine half time) was calculated by the next formula.
Acute Thyroxine Half-Time (time in minutes) = 45 log 2/log a -log b a= cpm of 1 ml. serum at 15 min. after intravenous injection of 20 micro C. I¹³¹- T₄
b = cpm of 1 ml. serum at 60 min. after intravenous injection of 20 micro C.I¹³¹-T₄
With this method we measured acute thyroxine half-time in patients with various thyroidal diseases. The results were as follows :
Euthyroid 88.0+11.2
Nontreated hyperthyroid 66.3±4.8 (p<0.01)
Treated hyperthyroid 82.3+7.4
Nontreated hypothyroid 120.4±7.3 (p<0.01)
Treated hypothyroid 98.8±4.9
Simple goiter 78.3+9.5
There was no correlation between this acute thyroxine half-time and PBI, RSU, RBCU. But the correlations of PBI, serum cholesterol, RSU, RBCU, BMR to s/p ratio were r=0.61 (p<0.05), r=-0.80 (p<0.05), r=0.86 (p<0.05), r=0.80 (p<0.05), r=0.78 (p<0.05).
Conclusively, these findings suggested that deiodination concerning thyroxine metabolism was accelerated in hyperthyroid and prolonged in hypothyroid, and these states were normalized with therapy.

View full abstract

Quantitative determinations of urinary 17-ketosteroids (17-KS) and 17-hydroxycorticosteroids (17-OHCS) were made in 17 males and 8 females before and for 7 days following major surgery.
The method for quantitative determinations of urinary steroids using mild hydrosis of glucuronide and sulfate followed by purification and fractionation with elution chromatography on Amberlite IRC-50 has been applied to urinary steroid analysis in order to obtain accurate changes of urinary 17-KS in moderate to major surgery. The excretion value of total 17-KS is shown in the sum of common individual 17-KS.
The following results were obtained :
1) The operation caused a marked rise in the excretion of total 17-OHCS. On the other hand, a significant decrease in excretion of total 17-KS was found following surgery.
2) Following the operation, the excretion of C₁₉O₂-17-KS significantly decreased, but the excretion of C₁₉O₃-17-KS in urine remained almost unchanged.
3) The significant decrease in the excretion of aetiocholanolone and androsterone was more marked in males than females suggesting a decreased testicular function following surgery.
4) The ratio of aetiocholanolone to androsterone increased post-operatively.
From these results, it can be suggested that these findings of urinary steroids following the operation may reflect some nonspecific metabolic responses to major surgical stress.

View full abstract

The present report describes a two antibody radioimmunoassay technique utilizing ¹²⁵I-HCG which permits determination of HLH in unextracted small urine samples.
Antisera to HCG were prepared in rabbits by injecting 2 mg of HCG (5,427 IU/mg). Anti ovine LH sera were obtained from rabbits by injecting 4 mg of NIH-LH-Sll. This was repeated 5 times weekly. Four kinds of precipitating antisera were employed ; goat anti rabbit gamma globulin serum (anti-RGG), sheep anti rabbit globulin serum (anti-RG), goat anti-RGG serum absorbed with Fab and guinea pig and rabbit globulin serum.
HCG (12,000 IU/mg), kindly donated by Dr. P. Donini, and NIH-LH-Sll were labeled with ¹²⁵I or ¹³¹I based on the method of Greenwood et al. Specific activities of 50-150 μc/μg or 250 μc/μg were obtained.
Figure 3 shows the essential features of the assay method used in these experiments. This radioimmunoassay procedure for LH utilizes a technique similar to that described by Hales & Randle for Insulin.
For the standard of HLH, 2nd-IRP-HMG, kindly provided by Dr. D.R. Bangham, was used. The diluent consists of 0.5% bovine serum albumin in a sodium phosphate buffer.
Experiments were designed to study the parameters of this method. The following results were obtained.
1) The particular anti-HCG serum selected for use in the assay was the one which gave the largest reduction in bound radioactivity when unlabeled standard hormone was added to an assay mixture.
2) Goat anti-RGG serum (absorbed with Fab) and goat anti-RGG serum gave high recoveries at lower concentration. An appropriate dilution of anti-RGG serum to anti-HCG serum was selected to work in the region of anti-RGG serum excess.
3) If an anti-HCG serum was used at a low dilution, then smaller quantities of labeled HCG was chosen in the condition of the anti-HCG serum excess (Fig. 8).
¹²⁵I-HCG lost their reacting capacity with anti-HCG serum after 2 months (Fig. 8 dash line).
4) Anti-HCG serum did not react with NIH-LH-Sll, but HCG cross reacted partially with anti-ovine LH serum (Fig. 4 dash line).
5) Increasing the time of centrifugation at room temperature resulted in slightly larger per cent precipitated. The increase was marked especially when the concentrations of the standard were low and polyethylene tubes were used as reacting tubes.
6) The optimum preincubation time was found to be 24 hr. and over.
7) By prolonging the assay incubation, the recovery was increased. It required 6-10 days for the incubations to reach equilibrium. Preincubation and incubation were carried out at 4°C. The incubation time to reach equilibrium was shorter at 37°C than 4°C (Fig. 13).
8) There was an optimal volume for the reaction mixture. A 0.2 ml of assay samples was used (Fig. 14).
Standard curves for HMG preparations and HCG preparations in a typical assay are shown in Fig. 15. The two groups of standard curves were parallel.
The sensitivity of this assay system is influenced by changes in dilution of anti-HCG serum. Anti-HCG serum used at a dilution of 1 : 10,000, the system has a sensitivity of about 5 mIU/ml.
To evaluate reproducibility and accuracy, the same urine samples were analyzed in 5 replicate assays. Mean, standard deviation and coefficient of variation (standard deviation divided by the mean) are shown in Table 1. The mean coefficient of variation measured for 8 urine samples containing different LH levels were 20.9 per cent.
The index of precision (λ) for the straight portion of standard curves from ten consecutive assays varied from 0.010 to 0.065 (mean=0.033±0.017 SD).
Moreover, the recoveries tested by the addition of four 2nd-IRP concentrations (320, 80, 20 and 5 mIU/ml) to the same urine was 89.2,101.3,130 and 120% respectively, as shown in Fig. 17.
Urine samples and the standard were diluted in 1% BSA buffer or urine from children.

View full abstract

Urinary neutral steroid glucuronides in normal persons and in patients with various diseases were analysed by gas-liquid-chromatography with the method reported by E.H. Horning et al (1967).
1/20 of daily urine volume was hydrolyzed with β-glucuronidase and extracted 3 times with 1.2 volumes of ethyl acetate. Extract was washed with cold 2N-NaOH, acetic water and distilled water. MO-TMSi derivatives of extracted steroids were prepared by reaction with O-methylhydroxylamine hydrochloride and bistrimethylsilyl acetamide. 20μg of cholesteryl butyrate was added as a reference standard.
Analytical work was done by the temperature programmed gas-liquid-chromatographic procedure with OV-1 column and the use of flame ionization detection system. The results were as follows.
1) Recovery rate in extraction and preparing derivatives was 85.6±4.6% (Mean±S.D.).
2) Many neutral steroids were separated and identified well according to Methylen Unit Value or relative retention time to cholesteryl butyrate.
3) If responses of each derivative for cholesteryl butyrate are determined and peak areas of each derivative were corrected by peak area of 20 μg of cholesteryl butyrate, it was possible to measure individual steroids quantitatively.
4) Several fraction rates of steroid fractions measured from gaschromatogram in normal persons and patients with various diseases were the same as those that had been measured by other investigators with other methods.
5) Quantitative and qualitative variations of urinary neutral steroid glucuronides in adrenogenital syndrome, Cushing's syndrome, hypogonadism, hyperthyroidism, liver cirrhosis and collagen diseases were well observed with this method. Therefore this method is very convenient and useful to reveal abnormality in metabolism of steroid hormones and to diagnose several diseases.

View full abstract

The present studies were undertaken to investigate the influence of thyroid function on the metabolism of fatty acid and cholesterol in rats using acetate- 1 -¹⁴C and palmitate-1-¹⁴C. It was also thought of interest to ascertain whether thyroid stimulating hormone (TSH) affects lipid metabolism not only in the intact animal but also in the absence of the thyroid gland. Male rats of the Wistar strain weighing 140 to 150 gm were divided into 7 groups : hypophysectomized rats (operated 3 days prior to sacrifice), thyroidectomized rats (operated 7 days prior to sacrifice), intact rats given 30 μg of L-thyroxine per 100 gm of body weight daily for 5 days, thyroidectomized rats given 2.5 μg of L-thyroxine per 100 gm of body weight daily for 5 days, intact rats given 0.2 U.S.P.U. of TSH (Thytropar) daily for 7 days, thyroidectomized rats given 0.2 U.S.P.U. of TSH daily for 7 days and nontreated control. Each was then injected intraperitoneally with acetate- 1 ¹⁴C (5 μc per 100 gm of body weight) or palmitate-1-¹⁴C (1 μc per 100 gm of body weight). Following the administration of the isotope, liver, epididymal adipose tissue and blood samples were obtained at 0.25, 0.5, 1, 2, 6 and 24 hours. For the separation of fatty acids and cholesterol of tissues and plasma the method of Srere et al was used, and the cholesterol concentration was determined by the method of Abell et al. Radioactivities of these fractions were measured by a gas flow counter. Specific activity was expressed as cpm per mg of each fraction after correction for self-absorption.
1) Thyroidectomy resulted in an increase in the concentration of liver and plasma cholesterol, and L-thyroxine administration to thyroidectomized rats brought back this increase approximately to normal, whearas L-thyroxine administration to normal rats had no effect on the liyer and plasma cholesterol concentration. However, the concentration of cholesterol in epididymal adipose tissue was elevated by L-thyroxine treatment. The difference in cholesterol concentration in all these tissues between thyroidectomized rats and thyroidectomized rats given TSH was not significant.
2) In normal rats, the maximum acetate-1-¹⁴C incorporation for liver cholesterol and that for liver fatty acids occurred at 1 and 2 hours, respectively.
3) The specific activity of liver cholesterol 15 minutes after acetate-1-¹⁴C injection was much higher in intact rats given L-thyroxine than in nontreated control, while that 6 hours after injection was lower in the former than in the latter. Furthermore, in the thyroidectomized group the initial incorporation of acetate-1-¹⁴C into liver cholesterol was lower than nontreated control and this was restored by L-thyroxine administration, while the specific activity of liver cholesterol 24 hours after injection in the thyroidectomized group remained consistently high. These results demonstrate that thyroid hormone accelerates biosynthesis as well as degradation of cholesterol in the liver. Consequently, the increased concentration of plasma cholesterol observed in the hypothyroid state is probably explained by the differences in degree of the alteration of synthesis and elimination or destruction of cholesterol, i.e. the more marked decrease in the rate of elimination. It was also observed that hepatic cholesterogenesis was greatly reduced in hypophysectomized rats.
4) The administration of L-thyroxine, 30 μg per 100 gm of body weight daily for 5 days to produce hyperthyroidism, increased the initial incorporation of acetate-1-¹⁴C into liver fatty acids. Conversely, thyroidectomy resulted in a decrease in the hepatic lipogenesis. This impaired hepatic lipogenesis found in thyroidectomized rats, however, was not restored by L-thyroxine administration.

View full abstract