Urinary neutral steroids were showed the result of steroidgenesis and metabolisms on each organe in human. This study was carried out on fundamental experiment for method of the simultaneous extraction and separation for that urinary neutral steroids with good recovery and no destruction. According to the present experimental results, it was excellent that method of the hydrolysis was 2-step hydrolysis with β-glucuronidase hydrolysis and solvolysis, solvent of the extraction was β-glucuronidase fraction with ethyl aetate, sovolysis with ether, alkali wash was, cold 2N-NaOH, alkali and water wash was necessary the back extraction. Method for mass separation and purification of urinary neutral crude extract was excellent the florisil column chromatography. Method of the fractiontion was applicated for urinary 17-KS with TLC and GLC, for urinary 17-OHCS with TLC, for urinary cortol compouds with MOTOMORI's method, for urinary Pregnandiol and Pregnantriol with YOSHIDA's method. On this method, urinary Pregnandiol, Pregnantriol, 10 fraction of the 17-KS, 6 fraction of the 17-OHCS, and 3 fraction of the urinary Cortol compound was messured from one urinary extract.
Cortol compounds have been not determined as 17 OHCS or Porter-Silber chromogen, although they form one of the main groups of urinary metabolites of glucocorticoids. In this study, a new method for fractional determination of these three cortol compounds, and 17 OHCS in urine was established. The new method consisted of the following procedure. (1) Hydrolysis with, β-glucuronidase. (2) Extraction with ethyl acetate. (3) Separation of 1 fraction (17 KS and pregnandiol) and 11 fraction (17 OHCS, pregnantriol and cortol compounds) by first florisil column chromatography. (4a) Separation of 17 OHCS fraction by thin layer chromatography (solvent system: chloroform: methanol: water). (5a) Colorimetry as Porter-Silber chromogen. (5b) Separation of cortol compounds from other HIO4-17 KS steroids by second florisil column chromatography. (6b) Oxidation with periodic acid. (7a) Fractionation of three converted steroids by 6% water alumina column chromatogrphy. (7b) Colorimetry as Zimmermann chromogen. This procedures was cosisted from its fundamental experiments. Total recovery rate of β-cortol 100μg was average 66.2±2.0%. Identification of cortol compounds messured by this method was carried out on the Rf. values by TLC, RRT values by GLC, RRT values of TMSi-ether deliverts by GLC. 5β-pregnan-3α, 17α, 20, 21-tetrol named “11-deoxy-Cortol” was messured for excretion values in urine. Normal mean excretion values were as follow. Normal females (proliferative phase) THF 486±94, THE 1153±535, THS 256±57, (secretory phase) THF 457±58, THE 801±189, THS 261±46, normal males: THF 1789±360, THE 2741±568, THS 225±52, ug/day. Normal females (proliferative phase): Cortol 734.9±198.1, Cortolone 1120.6±241.2, 11-deoxy-Cortol 178.8±98.2, (secretory phase): Cortol 462.8±169.6, Cortolone 975.2±132.4, 11-deoxy-Cortol 207.6±58.9, normal males: Cortol 757.5±181.2, Cortolone 1514.9±227.3, 11-deoxy-Cortol 264.9±49.2 ug/day.
The way of preserving blood specimens is an important factor on the screening tests for acatalasemic gene carrier. Because, the decay time of catalase activity in blood depends on the physical conditions, largely on temperature, where the blood specimens are kept. The author investigated the decay rate of catalase activity under following conditions. 1. Preservation of human blood in a vacuum flask at the temperature of 010°C. 2. Preservation in a deep-freezer at -20°C. In the former condition the blood specimen did not lose catalase activity at least seven days, and afterwards decay went on gradually. On the contrary, in the latter frozen specimens some degree of decay occurred soon. Afterwards, however, they kept constant catalase activity, 84% of the initial value, for months. So, it is concluded that for the practice of screening tests preservation in a vacuum flask at the temperature of 010°C is preferable. Next, the author devised a handy, feasible way of screening tests named the !! floating disk method. !! It is a modification from M. Gagnon's estimation of catalase activity by measuring floating time of a piece of paper disk dipped with enzymatic specimen in hydrogen per-oxide solution. The screening level set up by this method is 12.5cm for the depth and 13.5 sec for the floating time.
Patients have more or less complains after the treatmaut for uterin cancer by irradiation or radical hysterectomy. In this series, 720 patients free from recurrence have been followed up and psychosomatic complain, urologic and bowel disorder, and sexual activity are analysed. The effects of hormonal treatment to those patients are evaluated.
β-glncuronidase, hydrolysate and solvolysate as the main enzyme group in the urinary hormones of steroids were measured and the enzyme work and the enzyme pattern as proposed by Yoshida of our laboratory were studied. The results are summarized as follows. 1. From the metabolic processes of urinary steroid hormones a simple method of estimating urinary steroid hormones was devised on the basis of the concept of the related enzyme work and enzyme patterns. 2. For the simple estimation two fractions. 17-KS and 17-KGS, as well as 17-OHCS, blue tetrozolium chromogen, pregnanetriol and pregnanediol to the total of 8 fractions were measured, and the work of related enzymes and the enzyme patterns were calculated subsequently. This method, when compared with the method of estimating 38 fractions, is found to attain fairly satisfactory results and it has been demonstrated to be applicable in clinical practice. 3. As for the enzyme work during the mestrual cycle 17-, 21-, 11-hydroxylated steroids and desmolated steroids all tended to be high before and after ovulation, low at the prolifetation stage, and somewhat high at the secretory stage. 4. As for the enzyme patterns during the menstrual cycle, the patterns of 11-and 21-hydroxylase both showed their peak in the middle of the proliferation stage, and by the desmolase pattern it increased somewhat before and after the ovulation. In the 17-hydroxylase pattern there could be observed no appreciable change. 5. As for the enzyme work during pregnancy the work of desmolated steroids and 17-hydroxylated steroids decreased in the third trimester of pregnancy, but the work of 11-and 21-hydroxylated steroids did not show any change due to pregnancy. 6. Regarding the enzyme pattern during pregnancy due to an acute increase of pregnanediol in the third trimester there chuld be observed a tendency of relative decrease in all these enzyme pattens. However, excepting those steroids producd and metabolized by feto-placental units such as pregnanediol and estriol, all the steroids-related enzyme group of maternal origin showed practically stablized enzyme work and pattern without any disturbance. 7. It is safe to say that this simple method of estimating steroid hormones in urine would serve sufficiently in the elucidation of pathogenesis, especially of the endocrinal diseases in future.
It is known that steroid hormones increase in the blood and urine during pregnancy. For the steroid metabolism in pregnancy there has been proposed a concept of feto-placental-maternal relationship by incubation experiments of fetal adrenals and placenta perfusion experiments and histochemical experiments. More recently with advent of isotopes it has become possible to measure blood steroid hormones so that the steroid metabolism in pregnancy is being elucidated by degrees. During the period from the terminal fetal stage to the newborn stage the adrenal cortex is known to undergo remarkable morphological changes. Namely, it is known that the fetal adrenal weigning ten times that of adult adrenal exists mostly in the fetal zone, which atrophies (shrinks) rapidly after birth and that the activity of 3β-ol-dehydrogenase in the fetal adrenal is extremely weak. In the fetal stage the steroid metabolism is conducted by the maternal-placental relationship, but on delivery this relationship is abruptly severed so that the steroid metabolism is carried out now independently. In view of a great morphological change of the adrenal and steroid metabolism in the fetal stage, the adaptation to the external environments in the newborn stage is truly mystical indeed. An especially interesting problem is just how 3β-ol-dehydrogenase known to possess only a weak activity in the fetal stage would operate in the newborn stage. For the purpose to elucidate how this 3β-ol-dehydrogenase operates on the urinary steroid hormones in newborn stage, the author paid a special attention to the urinary pregene-5-en-20α or β-diol (Δ5-pregnenediol to be abbreviated to Δ5Pd). Δ5-Pd is a saturated form pregnenolone and is a direct metabolite in the urine derived from pregnenolone. Consequently, Δ5Pd, when there occurs a decrease in the activity of 3-β-ol-dehydrogenase or some disturbance so that an excessive precursor flows into the pathway of steroid metabolism and the blood pregenenolone concentration becomes abnormally high, overflows resulting in a large quantity of it in the urine. In other words, when there is an excessive amount of Δ5Pd in the urine it can be considered that there is some abnormality of the steroid metabolism. Therefore, so long as the steroid metabolism in the newbron stage is being conducted smoothly, there would occur no appreciable change of Δ5Pd. With Δ5Pd as it is quite difficult to isolate and colorimetory, most of attempts of isolation-coloration procedures are done with Δ-5-3β-ol-steroid as a whole. The author devised a new method of isolation-coloration of Δ5Pd for the purpose to elucidate the adaptation of newborn to external environments from urinary steroid hormones, and carried out the measurements of Δ5Pd with lapse of time using urine collected from normal newborn babies. The results are summaried as follows. 1. By means of florisil column chromatography and impregnant TLC, it was possible to isolate Δ5Pd into pregnenolone, 17-OH-pregnenolone, 17-OH-progesteron and DHA, with allmost the same polarity. 2. By the new coloration method with Δ5Pd the color sensitivity as high as 1.5 to 2 times that of Oertel and Eik-Nes was attained. 3. As for the identification of Δ5Pd, it has been confirmed by the spots with the same Rf in the impregnant TLC, and in gas liquid chromatography using two kinds of filler by the same R. R. T., and by the coincidence of serial absorption curves in the coloration method. 4. As regards time-lapse (diurnal) changes of Δ5Pd, looking at the average values, there could be seen not so much change after birth, decreasing gradually. Looking at separate individuals, they could be divided into three groups: postnatal decreasing group, postnatal ascending group, and no change group.
With cases receiving extended panhysterectomy and those receiving (simple) total hysterectomy as the subjects of study, urinary steroid hormones were measured, and simultaneously the enzyme work and enzyme pattern related to the steroid metabolism, as proposed by Yoshida of our laboratory, were studied. The results may be summarized as follows. 1. Blue tetrazolium and 11-oxy-17-KGS In the cases receiving extended panhysterectomy the levels of these two substances in the urine on the day of operation did not reach so high levels as those in the cases undergoing total hysterectomy, but the values were significantly higher than those before operation, which gradually decreased on 2 to 4 postoperative days later. In the cases of total hysterectomy these values on the day of operation rose to a markedly higher level as compared with those before operation, but they rapidly recovered to preoperative values after two postoperative days and became stabilized. 2. 11-deoxy-17-KGS The level of urinary 11-deoxy-17-KGS did not show much change in both extended panhysterectomy group and total hysterectomy group. 3. 17-OHCS As to the level of urinary 17-OHCS, in extended panhysterectomy group some showed a somewhat higher value on the day of operation than that before operation, which gradually increased from two to 4 postoperative days but recovered rapidly by 6 postoperative days, while some did not show any change on the day of operation but a decreased value on the second postoperative day and recovered to the preoperative level after 4 postoperative days. In the total hysterectomy group the value on the day of operation was markedly higher that before operation in some cases, which rapidly decreased by 2 postoperative days but still higher than the preoperative level, thereafter decreasing gradually, recovered to the preoperative level by 6 postoperative days, while in others the level on the day of operation was somewhat higher than the preoperative level, which gradually increased from 2 to 4 postoperative days, and by 6 postoperative days it rapidly recovered to the preoperative level. 4. 11-deoxy-17-KS a) β-glucosiduronate In extended panhysterectomy group some showed high level of, β-glucosiduronate from the day of operation to the second postoperative day which recovered to the preoperative level by the 4th postoperative day, while others maintained a low level from the day of operation to the 4th day, recovering to the preoperative level on the 6th postoperative day. Total hysterectomy group showed a higher level of β-glucosiduronate than that before operation on the day of operation and recovered to the preoperative level after the second postoperative day which remained stable up to the 6th postoperative day. b) Solvolysate In extended panhysterectomy group there were some who showed no change in the level of solvolysate up to the 6th postoperative day while others who maintained a lower level than that before operation up to the 6th postoperative day. As to the total hysterectomy group there were some showing a higher level on the day of operation which recovered to the preoperative level on the sixth day, while there were others showing no change in the level for two days after operation but showing a higher level on the 4th postoperative day and recovering to the preoperative level on the sixth postoperative day. 5. 11-oxy-17-KS a) β-Glucosiduronate As to the changes of urinary β-glucosiduronate values in extended hysterectomy group there were some who showed a higher level for two postoperative days when compared with the preoperative level, which became stabilized after the 4th postoperative day, while there were some maintaining a stabilized lower level after operation.
Diagnostic value for ovarian tumor with Contact Compound Scope of the ultrasonic tomography is discussed. The size and localization of the tumor are relatively easy to determin with ultrasonic equipment but characteristics of the tumor, particularly benign or maligmant, has not been clarified. In this paper, tomographic patterns of vorious solid (Cystoadenocarcinoma, Granulosa cell tumor and Dysgerminoma) and cystic (Cystoadenoma and Dermoid cyst) tumors are demonstrated. Differential diagnosis to determin malignant or not was still difficult to obtain conclusive result but it was possible to diagnose solid one or not by the method.
For the assessment of androgenesity in vivo urinary 17-KS has often been mesured so far, but the significance of measuring 17-KS still poses many problems. On the other hand, the assessment of androgenesity on the basis of urinary testosterone is direct and superior, but on account of the close polarity of 17-KS fraction and gestagen on chromatography as well as because they are steroids secreted only in a minimal quantity, the only available method reported is to isolate them in three steps at least by paper chromatography or thin layer chromatography. In view of this, the author devised a new method to assay urinary testosterone. This method uses two-step hydrolysis; first β-glucuronidase hydrolysis and solvolysis. Then the extraction (1) with ethylacetate is carried out. Next, the isolation (2) is conducted by column chromatography, and the separation (3) by impregnant thin layer chromatography. After scraping off the separate, it is oxidized with chromic acid (4) and finally, the qualitative analysis is done by the coloration method of Zimmerman. By this method of isolation and qualitative analysis of testosterone from urine samples fairly satisfactory results were obtained. The results have demonstrated that the pattern of urinary testosterone during normal menstrual cycle shows its peak before and after ovulation as well as immediately before menses. In addition, testosterone in the urine also exists in the form of a solvolysate, and its pattern during normal menstrual cycle and its alerations on taking drugsshow behaviors often different from those of β-glucuronidase hydrolysate.
In order to evaluate the effectiveness of public health activities in the community, the methods of calculating crude death rate, standardardized or corrected death rate are available, for it is important to measure the effect from a specific disease to the population of the community and the goals of public health activities are, in general, to decrease deaths from specific diseases. However, these rates are not absolute indicators, because they are influenced by age distribution of the population and/or by the standardized population used. Life-table based on the death rate from a specific desease is of great importance, showing the influence of the disease on a certain population without any other death. The author has deviced a new method to calculate life-table by deaths of a specific disease, which is much easier than the methods by Lotka, Wiesler, Greville and Farr. Comparison between his new method and the others has been discussed, showing the examples of several specific diseases in our country.
The healing process of anastomosed jejunum of rats is studied using radioautography and electron microscopy. From three hours to seven days after operation, DNA synthesizing zone increases in the crypt cells around the anastomosed area. This phenomenon is not observed at later stages after operation. Regenerative epithelial cells start to appear from the 2nd post-operative day. They are cubic in shape and their micro-villi are shorter and less numerous than those seen in the matured epithelial cells. In the regenerative epithelial cells, one seldom sees endoplasmic reticulum in contrast to numerous free ribosomes. Parallel to the post-operative days, villous regeneration has begun to form themselves like budding and consequently regenerative cells become more differentiated, resembling the matured villous cells. In stroma around the anastomosed area, the numbers of fibroblasts rapidly increase at the 3rd post-operative day, and one observes extensive development and dilatation of endoplasmic reticula. Small numbers of collagen fibrils are found extracellularly at the 4th post-operative day, and numbers of fibrils and their widths have continued to increase as the time lapses. In malnutrition, DNA synthesizing zone in the crypt cells is on the lesser increase and the proliferation of connective tissue cells is in retard as compared with control animals. Fibroblasts show autolytic degeneration at the 7th post-operative day in the ill-nourished animals.
Histochemical and electron microscopic studies on the healing process in human gastric and duodenal ulcers were presented. The material for study was obtained from 83 surgically resected stomachs, including 68 patients with gastric ulcer and 15 with duodenal ulcer. These sections were stained with PAS, PAM, Alcian-blue and Toluidine-blue. One of the characteristic features of gastric and duodenal ulcer healing was the presence of simple regenerative epithelium advancing into on the lesion. They were generally low columnal cells, approximately 7μ wide. In these cells, the mitotic figure was occasionally present and “Undifferentiated regenerative cell” appeared. The gastric and duodenal ulcer resembled each other in the undifferentiated regenerative cell. These cells had short and wide microvilli. In these cells, one seldom saw the mitochondria, Golgi complex and mucin granules in contrast to numerous free ribsomes and glycogen granules. They gradually differentiated into the various kinds of the matured epithelial cell as a part from the center of the ulcer.
Human gall bladders specimens obtained soon after cholecystectomy from 50 patients were observed under electron microscopy. Four types of cells were distinguished by light and electron microscopy in the human gall bladder epithelium, i.e., “Ordinary epithelial cell”, “Clear cell”, “Dark cell”, and “Basal cell”. The Clear cell have been subdivided at least two types by electron microscopy. One was degenerative type and another was immatured one. It was also observed that the mechanism of discharge of secretory granules was the “Merocrine mode”. In the cholesterosis, it was assemed that cholesterol was absorbed into the epithelial cells from the bile and accumlated in their basal area, then discharged through their basis. It was taken by phagocytes which gradually transformed into the foamy cells.