Okayama University Medical School

Separation of both forms of the direct bilirubin were carried out from the dog's gallbladder bile, and further isolations of them were also done. 1. The natural salt-form bilirubin was isolated after separation on the column of aluminium oxide with a n-propanolic aqueous solution. 2. The natural salt-form bilirubin was obtained in amorphous yellow powders which were strongly hygroscopic and easily soluble in water and methanol but not in chloroform or carbon tetrachloride. An aqueous solution of these powders showed both the direct diazo and Gmelin reaction, but neither Ehrlich's aldehyde nor Schlesinger reaction. The salt-form bilirubin was transferred into chloroform only when some quantities of hydrochloric acid were added to a mixture of chloroform and an aqueous solution of it. 3. The absorption maxima of the natural salt-form bilirubin existed at 420 to 430 mμ in a methanolic solution and at 425 or 435mμ in 50% or 10% n-propanol. 4. The natural ester-form bilirubin was isolated after separating on the column of silica gel with a chloroformethanolic mixture. 5. The natural ester-form bilirubin was obtained in amorphous greenish yellow powders. It was further hygroscopic and easily soluble in water and methanol but not in chloroform or carbon tetrachloride. An aqueous solution of it showed the direct diazo and Gmelin reaction, but neither Ehrlich's aldehyde nor Schlesinger's reaction. No pigment was transferred into chloroform even if some quantities of hydrochloric acid were added to a mixture of chloroform and an aqueous solution of it, but did by saponification with 5% methanolic potash. 6. The absorption maxima of the natural ester-form bilirubin existed at 415 mμ in both methanolic and aqueous solutions.

Descriptions are carried on the method how to separate the indirect bilirubin from the chloroform extracts of the dried dog's gallbeadder bile by adsorption chromatography. 1. The optimal concentrations of the bilirubin content were 2 to 4 mg/100 ml when 1 ml of the sample was adsorbed on the Tswett tube of about 10 mm diameter. 2. Though several zones of the indirect bilirubin were separated on the column of silica gel when developed with various solvents, these zones were proved to be mingled with some oxidized or other intermediate products and the separation like this was thought to owe to the activity of the adsorbents. 3. The chromatogram of the crystalline bilirubin resembled to the one formed by the indirect bilirubin in the chloroform extracts. 4. The chromatogram of the chromatographically separated indirect bilirubin was similar to the former. 5. The absorption maxima of a chloroform solution of the natural indirect bilirubin existed at 450 mμ in the visible range, and it was the same as the maxima of the crystalline bilirubins.

1. Absorption maxima of hydrochloric biliverdins derived from the natural indirect bilirubin existed at 680 mμ and 375 mμ, but the maxima of biliverdins purified on the column of silica gel existed at 640 mμ and 390 mμ. 2. The natural salt-form bilirubin was oxidized by hydrochloric acid to biliverdin, of which absorption maxima existed at 685 mμ and 370 mμ in a methanolic solution as well as in 5% hydrochloric methanol, but the purified biliverdin in chloroform solution showed the maxima at 640 mμ and 390 mμ. 3. The natural ester-form bilirubin could be transformed into biliverdin by oxidation of its alcoholic solution in the presence of hydrochloric acid. The crude biliverdin had absorption maxima at 645 to 655 mμ, 600 mμ and 320 mμ, and the crude hydrochloric biliverdin had the maxima at 665 to 675 mμ, 620 mμ and in the near ultra-violet range, while the purified biliverdin in chloroform solution had the maxima at 640 mμ and 380 mμ. 4. The biliverdins derived from the indirect, salt-form and ester-form bilirubin had quite similar absorption maxima after purifications by adsorption chromatography.

Separation of the urinary ester-form bilirubin was attempted, and the results obtained may be summarized as follows: 1. A brown pigment was obtained from jaundiced urine by the following procedures; namely, salting out, methanol extraction, chloroform flocculation, and separation on cellulose column. The pigment has been found to be easily soluble in water, displaying the absorption maximum at 420 - 410 mμ at pH 7.0, and it also gave a positive reaction both to GMELIN's and EHRLICH's diazo reagents within a minute without the addition of alcohol. These characteristics agree well with those of the socalled ester-form bilirubin. 2. On the basis of the results of paper chromatography and paper electrophoresis, the pigment has been determined to contain no amino acid, steroid, nor reducing substance. Moreover, no glucuronic acid could be detected whether examined in vitro or by paper chromatography together with paper electrophoresis, either.

1) I designed a new micro-method for complement fixation test by means of a capillary pipette. 2) By this method, the complement-fixing antibodies in an individual mouse could be tested without taking its life. 3} The complement fixation titers in mice immunized with Japanese B encephalitis had a considerable individuality. 4) An adjuvant containing anhydrous lanoline and paraffin-oil, when mixed with Japanese B encephalitis vaccine, was effective to potent complement-fixing antibody productions in mice to this antigen.

The ongm and characteristics in transmission and morphology of spontaneous lymphatic leukemia in a low-leukemic strain C3Hf have been described. The leukemia line is being currently subjected to a vigorous search for the presence of a filtrable leukemia agent.

The incidences, distribution and histopathological findings of N, N'-dimethylnitrosourea (DMNU)-induced brain microtumors in Sprague-Dawley rats were studied. Subcutaneous injections of DMNU in young adult rats one a week resulted in the induction of 122 gliomas in 38 animals with an incidence of 69% after a time lapse of between 157 and 246 days from the first injection. Of these tumors, 66 were classified as microtumors (diameter less than about 1 mm) by detailed light microscopy observation of serial sections. The microtumors were of 3 types: 55 oligodendrogliomas, 8 astrocytomas and 3 mixed gliomas. As the tumors became larger in size, anaplasia appeared, especially in the central part of the tumors. The microtumors developed randomly throughout the brain. It was concluded that, in adult rat brains, the target cells of DMNU were well differentiated glial cells which had already migrated from the matrix layer.

Lysis of fibrin was first recognized by MORGAGNI in 1769, observing a liquid blood in a patient of acute death, and the phenomenon was named as fibrinolysis by DASTRE in 1893. In 1937, MACFARLANE recognized in a patient after cholecystectomy that the blood clot was lysed completely in the following morning. Since then, much attention has been paid clinically on fibrinolysis and it has been said to occur in case receiving a large amount of blood transfusion, shock, cancer, obstetric diseases, hemophilia, various drug poisonings, allergic diseases, after irradiation and after the operations of lung, pancreas and prostate. In our department, also, the similar phenomenon was recognized often in association with cardiac surgery using the artificial heart-lung machine, and a difficulty in hemostasis was encountered postoperatively. We have been studying, therefore, on fibrinolysis in open heart surgery.

To study autoantibodies against liver cell surface membrane clinically, anti-LP-1 and anti-Tamm-Horsfall glycoprotein (THGP) were determined in the sera of patients with various liver diseases. They were detected by ADCC assay using antigen-coated cells as the target. A high incidence of anti-LP-1 was seen in chronic hepatitis (CH), liver cirrhosis (LC), primary hepatic cancer with cirrhosis (PHC), and primary biliary cirrhosis. The incidence of anti-THGP was also high in CH, LC, and PHC. Both anti-LP-1 and anti-THGP were detected in 2 of 3 patients with lupoid hepatitis. The patients studied here had no obvious evidence of renal tubular acidosis or pyelonephritis. Serum alanine transaminase activity, serum gamma-globulin content, and the presence of rheumatoid factors were not associated significantly with the presence of anti-LP-1 or anti-THGP in chronic liver disease. In 7 cases of CH tested serially during their clinical course, anti-LP-1 and/or anti-THGP tended to appear during acute exacerbations. The demonstration of anti-LP-1 and anti-THGP suggested that their appearance was related to the development of chronic liver disease.

Two forms of the direct bilirubin separated from the dried canine cholecystic bile were subjected to paper chromatography and emission or infra-red spectroscopy, and the following results were obtained: 1. The two forms of the direct bilirubin contain plenty of bile acid or its salt, and benzidine- and ninhydrine-positive substances together with various inorganic elements were also detected. 2. The ester-form bilirubin had carboxyl radical by infrared spectroscopy. But it will not be easily concluded that an existence of carboxyl radical will owe to free carboxyl radical of the dibasic acid bilirubin by the reason why an existence of plenty of bile acid in the sample may inhibit the characteristic absorption of ester. 3. It may be suggested that the two forms of the direct bilirubin combine with bile acid or its salt, and that the affinity between them is stronger in the salt-form bilirubin. 4. It seems probable that properties of the salt-form and ester-form bilirubins are not influenced by an existence of bile acid or its salt, and further by acornbination with it.

For the purpose of obtaining the dibasic acid indirect bilirubin in a pure state from the dried canine cholecystic bile, an optimal developing solvent was selected by paper partition chromatography as a preliminary experiment, and it was isolated on cellulose column as an applied experiment. 1. The dibasic acid indirect bilirubin was separable at the starting point in a pure state by paper chromatography under development with the top layer of a n-butanol, acetic acid, water mixture (4:1:5). 2. The dibasic acid indirect bilirubin formed a fixed band at the upper starting place on cellulose column under development with the top layer of a n-butanol, acetic acid, water mixture (4:1:5), and no other substance could be detected there. 3. The dibasic acid indirect bilirubin existing in the fixed band could be eluted out into chloroform with a 1% acetic acid solution. An orange yellow powder was obtained from the eluate by evaporating the solvent in vacuo. 4. Thus separated orange yellow powder agreed well with the crystalline bilirubin in the solubility into organic or inorganic solvents and in the spectrochemical characteristics as well as in the chemical properties.

To study the mechanism of DNA excision repair, a DNA repair system employing permeable mouse sarcoma (SR-C3H/He) cells was established and characterized. SR-C3H/He cells were permeabilized with a 0.0175% Triton X-100 solution. The permeable cells were treated with 1 mM ATP and 0.11 mM bleomycin, and then washed thoroughly to remove ATP and bleomycin. Repair DNA synthesis occurred in the bleomycin-damaged, permeable SR-C3H/He cells when incubated with ATP and four deoxyribonucleoside triphosphates. The repair nature of the DNA synthesis was confirmed by the BrdUMP density shift technique, and by the reduced sensitivity of the newly synthesized DNA to Escherichia coli exonuclease III. The DNA synthesis was optimally enhanced by addition of 0.08 M NaCl. Studies using selective inhibitors of DNA synthesis showed that aphidicolin-sensitive DNA polymerase (DNA polymerase alpha and/or delta) and DNA polymerase beta were involved in the repair process. The present DNA repair system is thought to be useful to study nuclear DNA damage by bleomycin, removal of the damaged ends by an exonuclease, repair DNA synthesis by DNA polymerases and repair patch ligation by DNA ligase(s).

Cellular immunity against human bile proteins was investigated by the leukocyte migration inhibition test (LMIT) with 13 primary biliary cirrhosis (PBC) patients, 10 chronic aggressive hepatitis (CAH) patients and 21 healthy adults. Hepatic bile taken from patients operated on for lithiasis of the biliary tract was fractionated into five fractions with Sepharose 6B gel. A subtoxic dose of each fraction was determined in the healthy adults, and used as the antigen for LMIT. Out of the 5 fractions, only the third fraction led to an LMIT positive response in 8 out of 11 (73%) PBC patients and in 1 out of 10 (10%) CAH patients. The difference between PBC and CAH was significant (p less than 0.005). The remaining 3 PBC patients with LMIT negative responses were all under D-penicillamine treatment. Antibody to each fraction was prepared in rabbits. Using the antibodies after absorption with human serum, the localization of the antigens which were present in each fraction was investigated immunohistochemically using human liver sections. The antigen to the anti-first fraction antibody was detected specifically in the epithelial cells of the bile ducts and the ductules, and the antigen to the anti-third fraction antibody was detected specifically on the membrane of the bile canalicules. The third fraction was fractionated into three fractions by Sephadex G-200 gel. Only the first of the 3 fractions showed an LMIT positive response in 3 PBC patients, and its molecular weight was determined to be about 500,000. It is concluded that PBC patients develop cellular immunity against canalicular-antigen-containing fractions but not ductal-antigen-containing ones.

The effect of glucose load on the levels of blood glucose, serum non-esterified fatty acids (NEFA) and liver citrate was investigated in carbontetrachloride-intoxicated (injured) rats and compared with non-intoxicated controls. The citrate level in the liver from injured animals showed 15-fold of the value of the control. Glucose load on these animals caused gradual decrease in the citrate level, whereas similar administration to the control caused inverse results. The serum NEFA levels were lowered by glucose load in both of injured and control animals. The pattern of changes in the citrate level after glucose load in the liver from injured animals was similar to that in the muscle from the control, suggesting a similarity on citrate metabolism between the injured liver and the muscle.The possible mechanisms for these results were discussed in relation to the difference in citrate metabolism between the liver and the muscle.

For the purpose to study the citrate metabolism in liver diseases, blood citrate, blood glucose and serum non-esterified fatty acids (NEFA) in fasting state were measured in the subjects with chronic hepatitis and with liver cirrhosis. Citrate and glucose were measured by the enzymatic methods. NEFA was measured colorimetrically. Fasting blood citrate level was investigated in relation to the type and extent of these liver diseases.Results revealed the following: 1. Fasting blood citrate level rose with the severity of liver diseases, especially in decompensated liver cirrhosis. 2. No significant difference in fasting blood citrate level was found between the subjects with and without glucose intolerance. 3. Fasting blood citrate level had a closer correlation with serum NEFA level than with blood glucose level. From these results, it has been concluded that the increase in blood citrate level in liver diseases is due to the impaired uptake of citrate by the liver and the increased release of citrate from peripheral tissues.

By our method of bone marrow culture and peripheral leucocyte culture, the differentiation of leukemia from other diseases is simplified. By this method the acute form of leukemia can be differentiated from the chronic form, and the classification of leukemia by the leucocyte series becomes easy and exact. It is believed that this method is clinically quite useful.

Of eosinophilias that we often encounter clinically, we selected two of the most representative ones, namely, hookworm diseae and bronchial astma, for our present sternal bone-marrow tissue culture, and studied the movement patterns and wandering capacity of eosinophils. As the results, even in those eosinophils that show no significant change other than the increase in number in ordinary stained-smear specimens of peripheral blood or bone marrow, it has been clarified that, when observed under living condition, they reveal a picture specific to individualistic behaviors according to diseases. Therefore, it can be assumed that in the pathologic condition what is known as eosinopilia not only eosinophils increase in number but also qualitative changes of eosinophlils specific to each disease are brought about, and consequently these specific changes are reflected on the movement patterns of the eosinophil.

For the purpose to look into the regulatory mechanism of erythropoiesis, changes in the cell volume and the cell size of the erythroid cells have been observed in peripheral blood and marrow from normal and phenylhydrazine induced anemic rabbits. And the following results have been obtained: 1. After the injection of phenylhydrazine hydrochloride a hemolytic anemia can be induced with a marked increase in the reticulocyte number. The cell volume increases with the advance of anemia but it is never proportional to the increase of reticulocyte number. The MCV reaches the value twice the normal but it never exceeds the threshold. 2. In bone marrow the smaller sized orthochromatic cells are reduced extremely in number or obliterated in anemic animals. As there is not any marked difference in cell size of polychromatic erythroblasts between normal and anemic animals, the large red cells of anemic animal will be formed by denuc1eation of the polychromatic erythroblasts. 3. The percentage of basophilic erythroblasts is increased in anemic animal suggesting an accelerated differentiation-division of proerythroblasts to basophilic ones. 4. The data strongly support the denuc1eation at polychromatic stage in emergency but not at the younger stages than polychromatic erythroblast. Data also suggest that in severe anemia an accelerated cell division occurs, especially in the stage from proerythroblast to basophilic erythroblast.

For the purpose to reveal the mechanism of the stimulated erythropoiesis in anemic condition, the author observed the numerical changes of the erythroblasts from normal rabbit bone marrow cultured under the environment of varied oxygen tensions, and revealed the following: 1. The erythroblasts incubated with air are increased after 24 to 48 hours and decreased gradually disappearing by 120 hours with a corresponding increase of erythrocytes. But no active proliferation of the stem cells or proerythroblasts is observed, all the cells have differentiated to erythrocytes. Hyperoxygen tension suppresses the increase of erythroblasts slightly, while hypoxygen tension stimulates the increase. Data suggest that the cell number destined to be ineffective erythropoiesis is regulated by oxygen tensions of the environment. 2. Basophilic erythroblasts are reduced in number from the beginning showing not any increasing tendency. The reducing rate is almost the same among those cultured under the hypo- and hyperoxygen tension, comparable to that incubated with air. 3. The hypoxygen tension brings about a marked increase in the number of orthochromatic erythroblasts with a decrease in polychromatic erythroblasts suggesting an accelerated cell differentiation, while the hyperoxygen tension elicits the suppression in the formation of orthochromatic erythroblasts with suppressed differentiation. Data also show the lack of denucleation mechanism in polychromatic stages in vitro differing from the case of the bone marrow of anemic animal.

From these results it is but natural to assume that the antigen-antibody reaction is involved in the phenomenon, eosinophilia. The antigen in this instance is the filtrate of hookworm emulsion, and the serum of hookworm disease as well as the bone marrow can be thought to contain the antibody. In any case, so long as the medium contains the serum or bone marrow or both of them obtained from the patient of hookworm disease, eosinophilia and the acceleration in the motility of eosinophils are brought about in the growth zone by addition of the filtrate of hookworm emulsion. Therfore, as for the mechanism inducing hookworm eosinophilia, it may by interpreted that the patient of hookworm disese is repeatedly sensitized by the antigen arising all probability from the metabolic products of hookworms or from the dead bodies of the worms; and producing the antibody in tissues and blood, thus the antigen-antibody reaction is elicited in vivo as long as hookworms live in the human body so that the increase in the mitosis and the acceration in the motility of eosinophils in the bone marrow are brought about with the resultant continuous discharge of a large quantity of eosinophils from the bone marrow parenchma into the sinusoids, there by inducing eosinophilia in the peripheral blood.