In reference to microdetermination of catecholamine, our efforts in the past few years have been concentrated on estimation of free catecholamine in urine or plasma, however the detection has so far met with certain difficulties.
Recently, Armstrong reported that 3-methoxy-4-hydroxymandelic acid was excreted in urine as a major metabolite of epinephrine and norepinephrine. Following this, some methods for estimation of 3-methoxy-4-hydroxymandelic acid in urine have presented.
We have followed Sandler's method without delay and partially modified as well as simplified the entire procedure and we have found it diagnostically significant in clinical application.
In brief, we employed the “omitting of indol reagent” -technique as a blank preparation. We invite your suggestion for our modification.

View full abstract

The author has attempted to establish basic experimental conditions suitable for metabolic studies, including bioassay of insulin-like activity of blood, of epididymal fat pad excised from Wistar strain rats. Ca. 100-120gm. weighing rats were used for the experiments. Epididymal adipose tissues were incubated by means of Warburg's apparatus in Krebs-Ringer's bicarbonate solution. Glucose and vorious hormones were added to the media and tested for their effect upon glucose uptake and QCO₂.
In the first step, the experiments were made on the comparative studies of different tissue sections of the epididymal fat pad with regard to their influences upon glucose uptake, QCO₂ and QO₂. A trisection pooled method as described in the figure was found to give most constant results, hence, this method was used throughout the whole experimental series. For the standard metabolic response of epididymal fat pad, glucose uptake and QCO₂, particularly the glucose uptake, were chosen.
Using this trisection method, insulin, glucagon, ACTH, cortisone acetate, sodium fluorate and malonic acid were tested for their effects on the glucose uptake and QCO₂ of the incubating adipose tissues.
As regards to the insulin effect, detailed studies were carried out to build up a bioassay method of minute insulin dose added to the incubation medium. ΔGlucose uptake and QCO₂ of incubated adipose tissues, particularly of the former, showed variations corresponding to the insulin dosage added to the medium, indicating dose-response relationship. Thus a bioassay method of minute insulin dosage could be deduced by the determination of Δglucose uptake from the incubation medium.
Using this bioassay method insulin-like activity of normal subjects and diabetic patients were determined in comparison with the results of rat diaphragm method.
The results are summarized as follows :
1) It has been proved that the isolated epididymal fat pad of rat is a suitable model for the study of carbohydrate metabolism of the tissues as in the case of the rat diaphragm. The glucose uptake and QCO₂ of the isolated epididymal fat pad responded quite well to insulin addition. Glucagon acted similarly but gave a mild effect upon glucose uptake and QCO₂, whereas the pituitary and adrenocortical hormones exhibited no apparent effect. Sodium fluorate and malonic acid apparently inhibited glucose uptake and QCO₂ of fat pad. Insulin effect upon epididymal fat pad could be accelerated by glucagon. But the effect was markedly suppressed with ACTH, cortisone acetate, sodium fluorate and malonic acid, particularly of the latter two.
2) A relation corresponding to linear regression was confirmed between the Δglucose uptake from the incubation medium and the log dose of insulin added to the medium. This insulin effect was observed with an insulin concentration up to 10^-5unit/ml, thus making it possible to determine quantitatively the insulin-like activity of serum in terms of Δglucose uptake. The mean insulin-like activity of serum in 13 normal subjects was 3.72±2.97 × 10^-4unit/ml. The mean insulin-like activity of serum in 12 diabetics was 2.66±2.18×10-4unit/ml.
3) In the comparative studies, the rat diaphragm showed much more basal glucose uptake than epididymal fat pad, whereas the latter exhibited much more 4glucose uptake expressed as insulin effect. These results seem to point upon a higher sensitivity of epididymal fat pad in the bioassay of insulin-like activity.
4) Insulin was added to normal or diabetic serum and their insulin effect was examined to see how the effect of exogenous insulin is influenced by human serum. In the case of normal serum,

View full abstract

Increased knowledge of metabolic disorder of adipose tissues in diabetes mellitus is presenting an important problem in the pathological physiology of diabetes. Stetten and Boxer (J. Biol. Chem., 156 : 271, 1944) studied the fat metabolism of diabetes using D₂O and reported the possible inhibition of fat synthesis from glucose in diabetic animals, resulting in glycosuria caused by the unused glucose. The direct metabolic studies of the isolated adipose tissue thus makes an interesting problem.
Studies were made on the behavior of glucose uptake and glycogen synthesis of the isolated diaphragm and liver slices of rats, and compared with those of epididymal adipose tissues with reference to normal and alloxan diabetic rats. Various hormones and antidiabetic drugs were also tested for their effect on the metabolism of isolated tissues excised from normal and diabetic rats. For the entire procedure, Warburg's apparatus was employed and the isolated tissues were incubated under ordinary experimental conditions, following the basic method previously described by Tsuji (Diabetes, Tokyo, 2 : 15, 1959.) and Waimatsu (will be published in Folia Endocrinologica Japonica). The results are summarized as follows :
1) Cortisone and prednisolone showed definite inhibitive effects upon glucose uptake of the epididymal adipose tissues. This inhibition could not be overcome by insulin. Comparative studies on the diaphragm showed only a slight increase of glucose uptake ; those on the liver slices showed a decrease of glucose uptake both by pretreatment with cortisone or prednisolone.
2) Pretreatment with ACTH gave decreased glucose uptake in the liver slices, but with the epididymal adipose tissues and diaphragm, both glucose uptake and insulin effect were found to be increased. As regards to epididymal adipose tissues it is assumed that ACTH can exert a direct lipolytic action, through which liberation of NEFA and increased glucose uptake could be induced. Similar effects on glucose uptake had been observed on pretreatment with growth hormone. After Waimatsu, however, in a certain pre-exhaustion state, the glucose uptake of epididymal adipose tissues seems to be accelerated. These variable glucose uptakes of epididymal adipose tissues seem to indicate that glucose uptake of this particular tissue is generally increased in the physiological conditions, when accompanied with moderate fat consumption.
3) Gonadotropin, androgen and estrogen were not capable of inducing appreciable metabolic changes of epididymal adipose tissues.
4) Isolated tissues of alloxan diabetic rats, in general, showed decreased glucose uptake, especially with the epididymal adipose tissues, but the insulin effect in vitro was markedly increased. This phenomenon implies the relatively easy restoration of the disturbed carbohydrate metabolism of experimental diabetic animals. Comparative therapeutic studies with insulin, D860 and DBI disclosed almost identical therapeutic effects with insulin and D860, where as DBI induced a decrease of glucose uptake after a long-term administration.
5) Formation and liberation of NEFA in and from the epididymal adipose tissues of alloxan diabetic rats showed an increase in proportion to the decreased glucose uptake. These metabolic disturbances of epididymal adipose tissues could be restored more or less by addition of glucose alone or glucose plus insulin to the incubation medium or through pretreatment of diabetic rats with insulin or D860. Addition of ACTH or hydrocortisone to the incubation medium invited further inhibition of glucose uptake and more liberation of NEFA possibly through the disturbance of fat synthesis. Metabolic behavior similar to this tendency was observed also in vivo experiments. Comparison of metabolic effects of insulin and D860 showed almost no difference in the above experiments.

View full abstract

Aceton dry powder of pig's anterior hypophysis were extracted with 2% saline water, and were centrifuged. The supernatant fluid was fractionated by continuous paper-electrophoresis (Condition : 26mA/35cm. 500v/40cm, ionic strength 0.05, Phosphoric buffer pH 8.5).
Each fraction was biologically assayed ; TSH activity by Greenspan's method, Gonadotropic activity by Florsheim's method, STH activity by Tibia-Test, and ACTH activity by Sayers-Munson's method. The fraction in the middle, TSH activity was clearly detected with slight gonadotropic activity. Other activities were not accounted. Contents of the fraction, in which TSH activity was revealed, were ultracentrifuged. The sedimentation curve was monophasic and the sedimentation constants was 2.28. Thus the molecular weight was calculated as about 20000.
In phosphate buffer solution, pH 4.5, the extracted substances of aceton dry powder of pig's anterior hypophysis were separated by continuous paper-electrophoresis into six fractions. In this case, as it was anticipated on account of acid solution thyrotropic activity would be diminished and only gonadotropic activity would be remained, the gonadotropic activity was measured by weighing the ovaries, uterus, prostate and seminal vesicle of hypophysectomized immature rats and the ovaries were examined histologically. On the second and the third fraction, which are numbered from anod, gonadotropic activity was found. The LH action was recognized in the second fraction and the FSH action in the third fraction.
NIH-FSH and NIH-LH which were introduced by Dr. Wilhelmi were also separeted by paperelectrophoresis, then NIH-FSH is separeted into two fraction, but NIH-LH separeted into only one fraction, so the purity of NIH-LH was confirmed.
Anteron was injected into hypophysectomized rat intravenously, ten minutes later blood was taken. The serum separeted by continuous paperelectrophoresis were examined for gonadotropic activity of each fraction by the method of weighing mouse uterus.
On gamma-globulin in the serum of rat, gonadotropic activity was recognized.
Crude gonadotropic substances of pregnant woman's urine and postmenopausal urine obtained by kaolin absorption method, were separeted into some fraction by continuous paperelectrophoresis (Condition : 26mA/35cm, 450v/40cm, ionic strength 0.05, phosphate buffer solution, pH 4.5).
Crude substance of postmenopasal woman's urine was separated into 3 fractions. Only in the first fraction, gonadotropic activity was revealed ; its FSH-action was very strong, but its LH-action was very weak.
Crude substance of the pregnant woman's urine in the former half term was separated into four fractions. Gonadotropic activity was recognized in the first and the second fraction ; The LH-action of it in the second fraction was stronger than that of the first fraction and FSH-action in the first fraction was stronger than that of the second.
Crude substance of the pregnant woman's urine in the later half term was separated into three fractions. In the first and second fraction of it gonadotropic activities were found, but FSH-action was a little weak comparing with that of the gonadotropic activity of the urine in the former half term. Even in the case of human chorionic gonadotropin and postmenopausal gonadotropin, LH-action and FSH-action were not separated perfectly.
1) Differences by paperelectrophoric condition.
TSH activity exists only in the middle fraction fractionated in basic solvent, but in acid solvent thyrotropic activity is apt to be lost. On the other hand, gonadotropic activity was not lost even in acetic solvent and in basic solvent. Therefore, for separation, extraction and purification of TSH, any procedure under acetic solvent are not desirable.
2) Thyrotropic substances and gonadotropic substances in serum.
Both TSH activity and gonadotropic activity were recognized on gamma-globulin.

View full abstract