Bile was collected with a duodenal tube from 14 controls and 45 cases with various liver diseases which were consisted of 8 cases with acute hepatitis, 10 cases with chronic hepatitis, 8 cases with cirrhosis of the liver, 4 cases with Gilbert's syndrome, 3 cases with Dubin-Johnson's syndrome and 12 cases with cholecystopathy. Bilirubin was extracted from the bile and separated with Billing's siliconized Kieselguhr column chromatography. Bilirubin-phosphate fraction showing positive phosphate ester reaction reported by Kondo, was fractionated with cellulose column chromatography as reported by Monobe, and finally its percentage to total direct bilirubin was calculated. Correlations between bilirubin-phosphate fraction and various liver function tests were studied and the following results were obtained. 1) Bilirubin-phosphate fraction was found to be 7.3% in cirrhosis of the liver, 5.5% in constitutional jaundice, 5.0% in acute hepatitis, 3.5% in chronic hepatitis, 2.2% in cholecystopathy and 2.1% in controls. Percentage of bilirubin-phosphate fraction was significantly higher in cirrhosis of the liver than in control, cholecystopathy and chronic hepatitis, and in acute hepatitis than in control and cholecystopathy. 2) There were good correlations among percentage of bilirubin-phosphate fraction, colloidal reaction (ZnTT, TTT) and serum γ-globulin fraction. 3) No significant correlations could be found among percentage of bilirubin-phosphate fraction, serum transaminase (GOT, GPT), cholesterol and alkaline phosphatase. 4) There was a good correlation between percentage of bilirubin-phosphate fraction and BSP retention at 45 minutes. 5) A significant negative correlation was obtained between percentage of bilirubin-phosphate fraction and KICG.
Non bilirubin fraction which was extracted from bile of various liver and biliary tract diseases and prepared from crystalline bilirubin after irradiation by fluorescence was studied. Following results about the nature of non bilirubin fraction positive for diazo reaction and its clinical significance was obtained. 1) The material obtained in the fraction was yellowish and soluble in water and showed positive diazo reaction. Its absorption maximum showed 545-555nm in water solution (pH 2.4) and unchanged by acidulation. 2) In thin layer chromatogram of this azo pigment of non bilirubin fraction, two to four spots appeared usually. This results suggested that it was not composed from one substance. The Rf values were different from those of already reported azo pigments in thin layer chromatogram and it was confirmed that the Rf value of one of these spots was changed by methylation. 3) Percentage of non bilirubin fraction in total bilirubin in bile of various liver and biliary tract diseases was significantly increased in comparison with that of control subjects. This ratio increased in the order of the group of constitutional jaundice, cirrhosis of the liver and chronic hepatitis respectively. 4) Significant correlations between percentage of non bilirubin fraction in total bilirubin and liver function tests (ZnTT, TTT, γ-globulin) were obtained. These findings suggested that non bilirubin fraction was increased in the case of hepatic parenchymal damage or the disturbance of the bilirubin conjugation enzyme.
The appearance of circulating antibodies to nuclear components (e.g. DNA, Histone, Nucleoprotein) is characteristic of SLE and may have pathogenic importance. But, it is not evident why these autoantibodies are produced. Concerning these immunological abnormalities, most research has concentrated on the humoral immune responses made against nuclear antigens. The role of cell-mediated immunity to unclear antigens in SLE has not been investigated. Therefore, the evidence of cell-mediated immunity to native calf thymus DNA was determined by MIT and LMIT in patients with SLE and normal subjects. The mean percent migration of MIT in SLE and normal subjects was 79.8±24.1 and 101.4±15.3%, respectively. The percent migration below 70.8% was considered to be a positive test and 17 out of 42 patients with SLE had a positive test. In contrast, none of normal subjects did. These findings indicate that there is cellulay hypersensitivity to native DNA and peripheral lymphocytes of SLE patients produced a migration inhibitory factor (MIF) with responding to it. No correlation could be made between results of MIT and staining patterns of antinuclear factor (AMF), titers of anti-DNA antibody, serum complement levels and activity of the disease. The results of LMIT was differed from those of MIT. The mean percent migration of LMIT was not significantly different in SLE patients and normal subjects, and only a few cases in SLE exhibited a positive test. Considering the dissociation of these results in MIT and LMIT, the conclusion has been made that MIT is more useful in investigation the existence of cellular hypersensitivity. In addition, the role of T cells in the development of autoimmunity was discussed in accordance with the existence of cellular hypersensitivity to native DNA in SLE.
The inflamed synovium of a patient with RA is intensively infiltrated with lymphocytes which often form follicles with aparent germinal centers. The synovial fluid contains aggregates of IgG and its complexes with rheumatoid factor. These evidences point out the immunological significance for the synovial inflammation and also suggest the need to determine whether cell-mediated immunity is concerned with the pathogenesis of RA. In the present study, an attempt has been made to determine whether heat-aggregated IgG and synovial crude extract have acted as antigns capable of causing cellular hypersensitivity in patients with RA. MIT and LMIT have been taken as in-vitro expression of cellular hypersensitivity. The value below the mean percent migration of normal subjects minus 2 SD was considered to be positive. MIT with heat-aggregated IgG was positive in 6 out of 14 patients with RA. Heat-aggregated IgG and synovial crude extract inhibited leucocyte migration in 4 out of 12 patients with RA. Indirect LMIT with heat-aggregated IgG was positive in 4 out of 14 patients with RA and with synovial crude extract in 5 out of 14 patients. None of normal subjects showed positive leucocyte migration inhibibition to both antigens. There was no correlation between positive tests and titers of rheumatoid factor, duration, activity of the disease. The data presented above raise the possibility that cell-mediated immune mechanisms play a role in the pathogenesis of RA.
Non-bilirubin fraction was extracted from the bile obtained from Wister strain rats and heterozygote Gunn rats, and separated by Ostrow1s method with minor modification. Azo-pigments was prepared with the non-bilirubin fraction after addition of Ehrlich1s diazo reagent. Significance of non-bilirubin fraction showing positive diazo reaction (NBAPF) was studied on the basis of the relation between the percentage of NBAPF to total bile azo pigments (proportion of NBAPF) and molar ratio of glucuronic acid to the ester-form bilirubin. The following results were obtained; 1) Averaged proportions of NBAPF were 0.94% in Wister strain rats and 2.50% in heterozygote Gunn rats, respectively. The latter proportion was higher than the former but not significant due to the wide variation of the measured values of the latter group. 2) Good correlation between the proportion of NBAPF and the molar ratio of glucuronic acid to the ester-form bilirubin was observed. 3) No significant correlation was observed between the bilirubin concentration in the bile and the molar ratio of glucuronic acid to the ester-form bilirubin or the proportion of NBAPF. 4) These results suggest when the conjugation of bilirubin and glucuronic acid in the liver was impaired, both the excretion of dipyrryl substance yielded from bilirubin and other conjugation mechanism increase compensatory.
Serum γ-glutamyl transpeptidase (γ-GTP) activities in patients with various hepatobiliary diseases were studied to elucidate the clinical significance of the enzyme. In an early stage of acute hepatitis, an elevation of the enzyme activity was slight or moderate, and the alteration of the activity in the clinical course behaved in a parallel manner with that of serum GPT activity. Estimation of the γ-GTP activity in a convalescent stage of acute hepatitis was useful for predicting the prognosis of the disease. A marked elevation of serum γ-GTP activity was observed in icteric primary hepatoma, especially when the tumor was large and situated at the hilar portion or in the right lobe of the liver, and in secondary carcinoma, the activity higher than that in primary hepatoma was obtained, when the cancer metastasized to the porta hepatis or infiltrated into the liver along the bile ducts even though jaundice was not revealed. In biliary diseases, the level of serum γ-GTP had a close correlation with those of alkaline phosphatase and leucine aminopeptidase.
For the purpose to elucidate the formation and the mechanism of lipid peroxidation products in the serum following irradiation, rabbits were irradiated once withe 1000R over the whole body, and serum lipid proxidation products as well as proxidation products in each lipid fractions were measured as TBA level. In addition, a study was made on the correlation between the peroxidation product and the change of fatty acids, and the following results were obtained. 1) The whole lipid content of serum: The whole lipid content was found to have increased about 2.6 times that before the irradiation by 24 post-irradiation hours, and even after the lapse of 48 hours such a tendency persisted. 2) Serum whole TBA level: By 24 post-irradiation hours the whole TBA level increased markedly up to about 6.5-fold that before the irradiation. 3) Lipid content of fraction: Except for cholesterol, every lipid fractions are found to have increased after irradiation. Especially marked is the increase of triglyceride. 4) TBA level of fractionated lipid: There is seen a marked increase of cholesterol ester, which proctically occupied entire serum TBA value. Next marked was the increase of phospholipid, and quantitatively it was classified that the increases seen in triglyceride and free fatty acids are not concerned with the rise in the free fatty acid content and TBA level. 5) Serum lipid contents and TBA level infasting: By taking the level of serum lipid 24 hours after the start of fasting as one, the serum lipid levels were studied at 48 and 72 hours after the start of fasting, and it was found that both serum lipid and TBA levels rose only very slightly. 6) Changes of fatty acids: The relative ratio of palmitic acid of the whole fatty acids increased after irradiation, and the ratio of linolic acid and linolenic acid was decreased by irradiation while by 48 hours the relative ratio of linolic acid was decreased to about 1/5 that before irradiation, and the relative ratio of linolenic acid was markedly decreased to about 1/35.
In order to elucidate which is the major functional unit, tubulus or glomerulus, on bilirubin excretion into urine, toad (Rana Clamitans) was used for the experiment because the distribution of blood vessels in toads was anatomically well separated to glomerulus and tubulus. 3H-bilirubin was prepared with Wilzbach's method. Crude direct 3H-bilirubin was extracted from the rat bile after injection of 3H-bilirubin dissolved in rat serum into the duodenal canal. Crude direct 3H-bilirubin were fractionated into three bilirubin fractions named indirect, salt-form and ester-form bilirubin by Kosaka-Hara's method. Change of excretion rate of ester-form bilirubin into urine in various blood pH ranged from 6.4 to 7.4 was investigated from the point of bilirubin-protein binding. The results were as follows; 1. 3H-radio activity in the urine was bound with the indirect bilirubin at the rate of 85.78% in average and with the ester-form bilirubin at the rate of 93.81% in average. 2. Ester-form bilirubin was excreted into the urine 1.69% in average by loading it to the renal portal vein, in the other hand, that of indirect bilirubin was 0.57% in average, in the first 5 minutes after loading. 3. Loading bilirubins to the renal artery, ester-form bilirubin was excreted into the urine, 10.60%, in average, indirect bilirubin was 0.08%, in average. 4. Excretion of ester-form bilirubin increased according to lowering blood pH, and reached maximum at pH 6.8. 5. As the results, most parts of bilirubin excreted into the urine was the ester-form bilirubin. The site of excretion of bilirubin was mainly tubulus and its rate of excretion might depend on affinity between serum protein and bilirubin.
In order to study the clinical significance of urinary bilirubin phosphate fraction, eight cases with acute hepatitis, seven cases with chronic hepatitis, five cases with liver cirrhosis, eight cases with obstructive jaundice and three cases with acute yellow liver atrophy were studied. The comparative study of the percentage of bilirubin phosphate fraction to the ester-form bilirubin and various liver function tests was investigated. Crude direct bilirubin was extracted from the jaundiced urine in the cases of liver diseases and fractionated into ester-form bilirubin by Kosaka-Hara's method and fractionated into three bilirubin fractions by the cellulose powder column chromatography with the solvent system of n-butanol, ethanol and water (4:1:2). The third fraction showed positive phosphate-ester reaction (bilirubin phosphate), and the glucuronic acid was negative in this fraction. 1. Percentages of bilirubin phosphate fraction to total ester-form bilirubin were 26.7 in the acute yellow liver atrophy, 11.27 in liver cirrhosis, 9.24 in the obstructive jaundice, 6.83 in the chronic hepatitis, and 6.09 in the acute hepatitis in each average, respectively. 2. Significant correlation could be found between the percentage of bilirubin phosphate fraction to the ester-form bilirubin and various liver function tests, that is, thymol turbidity test, zinc sulfate turbidity test and the percentage value of serum γ-globulin. 3. The decreasing tendency of the percentage of bilirubin phosphate fraction to the esterform bilirubin could be indicated the improvement of the liver function tests and the course of the disease.
(1) Tympanoplasties were done on 956 ears (789 persons) in our clinic for the past 10 years. (2) 684 operated ears of ages 10 to 29 were 72% in age distribution. (3) In 956 operated ears, the primary operations were 633 and the reoperations were 323. The rate of success of the hearing gain after surgery was 48.7% in the primary operation and was 44.4% in the reoperation. (4) In the primary operation, the conservative type (Wullstein I and II types) was done on 60.5% and its rate of success of hearing gain after surgery was 61.9%. The rate of success of hearing gain after surgery was 29.5%of the radical type group (Wullstein III and IV types, and modified types). (5) In the reoperation, the conservative type group was done on 29.1%, and its rate of success of hearing gain was 73.0%. The rate of success of hearing gain was 31.3% of the radical type group. (6) As the materials forming the tympanic membrane in the primary operation, the meatal skin flaps were used on 41.3%, the temporal fascia grafts were 35.1%, the thigh skin grafts were 12.8%, the double grafting were 9.8%. The rate of success of hearing gain after surgery was from 55.3% to 48.4% in the temporal fascia graft, meatal skin flap and double grafting, and was 28.0%in the thigh skin graft. In the reoperation, the meatal skin flaps were used on 52.3%, the thigh skin grafts were 17.3%, the double grafting were 15.1%, the temporal fascia graft were 14.6%. The rate of success of hearing gain after surgery was 81.3% in the double grafting, 68.4% in the temporal fascia graft, 32.8% in the meatel skin graft, 28.6% in the thigh skin graft. (7) As the materials of columella of the modified III and IV types in the primary operation, the ossicles were used on 76.3%, the tragal cartilage were on 14.4%. The rate of success of hearing gain after surgery was 41.7% in the tragal cartilage, 27.1% in the ossicles. In the reoperation, the ossicles were used on 36.3%, the tragal caltilage were 40.8%. The rate of success of hearing gain after suergery was 46.2% in ossicles, 42.9% in the tragal cartilage. (8) The closed method was done on 44.7% in the primary operation and 8.4% in the reoperation. On the postoperative situation, the closed method were superior to the open method. (9) The operation technique was described, which was considered to be the most preferable at preset in our clinic.
1) Eleven guanidino compounds were successively isolated and determined in uremic serum and brain by liquid chromatography using the cation-exchange resin (LCR-2, Jelco) and selecting the proper buffer system for elution. 2) The concentration of total guanidino compounds, including arginine, taurocyamine, glycocyamine, and guanidinosuccinic acid etc., was 143±39nmoles/ml in normal human serum (BUN:7-20 mg/dl). 3) The concentration of total guanidino compounds was 296±101nmoles/ml in serum of uremic patient (BUN: 40-161mg/dl). The concentration of taurocyamine and guanidinosuccinic acid were significantly elevated and methylguanidine was detectable in uremic serum. 4) Experimental uremia was induced by bilateral ureteral ligation of rabbit. Forty-eight hours after ligation when blood urea level rose to 90-219mg/dl, the concentration of guanidino compounds, especially taurocyamine and guanidinosuccinic acid, significantly increased. 5) The concentration of total guanidino compounds in normal rabbit brain was 359±37nmoles/g and that of γ-guanidino-β-hydroxybutyric acid and γ-guanidinobutyric acid were much higher than in serum. At forty-eight hours after bilateral ureteral ligation, significant increment of taurocyamine and decrease of glycocyamine and arginine were noted. The trace amount of methylguanidine was detected. 6) Generalized tonic and clonic convulsion was observed in the rabbit three minutes after intracisternal injection of methylguanidine (1.5mg/kg b. w.). Seizure discharges were recorded on electrocorticogram of the rabbit and cat, and it was comfirmed that spike activity originated in hippocampus on depth recordings.
The nature of biochemically synthetic bilirubin sulfate was examined on detail, and bilirubin sulfate and bilirubin glucuronide were measured in bile of heterozygote Gunn rat and Wister strain rat. Further, after the intrvenous injection of 3H-bilirubin sulfate into heterozygote Gunn rats and Wister strain rats, the biliary excretion rate of bilirubin sulfate was studied in them. The following results were gained by these studies. 1) The absorption peak was 452 nm in the synthetic bilirubin sulfate solution, pH 2.2, and its azo pigment showed each peak absorptions at 535-545 nm in pH 2.0 and at 565-575 nm in pH 1.0. The molar ratio between bilirubin and sulfate in the band of thin layer chromatography showing an Rf of 0.26 was determined from 1.72 to 1.96 with a mean of 1.78±0.09 S. D.. These data suggest that this compound is bilirubin disulfate. 2) In the bile of Gunn rats and Wister strain rats, the molar ratio of glucuronic acid to esterfrom bilirubin was inversely proportional to that of sulfate radical to direct bilirubin (r=-0.74, p<0.05). This implies that the conjugation of bilirubin with sulfate increases compensatedly as compared to the disturbance of that of bilirubin with glucuronic acid. 3) The excretion ratios of 2 hours to 24 hours in the intravenous 3H-bilirubin sulfate loading were 54.2% in Gunn rats and 63.4% in Wister strain rats. This finding supports the concept that bilirubin sulfate is easily excreted in the bile. The biliary excretion within 24 hours following the administration of bilirubin sulfate was 40.4% in Gunn rats and 69.4% in Wister strain rats, and these excretion rates decreased in parallel with the decrease of molar ratio in bilirubin glucuronide (r=0.96, p<0.01). It is suggested that the ability to excrete bilirubin sulfate may be the limiting factor in the existence of defective hepatic bilirubin glucuronidation.
Clinical significance of the components of direct bilirubin in bile was investigated on the basis of the relation between bilirubin sulfate fraction in bile and serological liver function tests in various liver diseases. Forty-three examined cases were consisted of eight cases with cholecystopathy, seven cases with acute hepatitis, nine cases with chronic hepatitis, seven cases with cirrhosis of the liver, four cases with Gilbert's syndrome and eight normal subjects, and their bile were collected with duodenal tube. The sulfate radical in direct bilirubin was quantitatively determined by Weber & Schalm's method and the molar ratio of sulfate radical to bilirubin was calculated. The following results were obtained. 1) No difference was observed between male and female, and among the age of patients on the molar ratios of sulfate to bilirubin. 2) The molar ratio of glucuronic acid to ester-form bilirubin was inversely proportional to that of sulfate radical to direct bilirubin. 3) The mean molar ratio of sulfate radical to direct bilirubin was found to be 0.10 in normal subjects, 0.08 in the cases with cholecystopathy, 0.27 in chronic hepatitis, 0.39 in acute hepatitis, 0.51 in liver cirrhosis and 0.48 in Gilbert's syndrome. The each values of liver diseases was significantly higher than normal subjects, and no difference between normal subjects and the cases with cholecystopathy. 4) Significant correlation was observed between the molar ratio of sulfate radical to direct bilirubin and following items, that is, ZnTT, TTT and γ-globulin; but no correlation between the molar ratio of sulfate radical to direct bilirubin and S-GOT, S-GPT, serum alkaline phosphatase, serum bilirubin and BSP retention test. These results suggest that the sulfate conjugation of bilirubin was compensated as compared to the disturbance of glucuronide formation of bilirubin in liver parenchymal damage.