In this paper, the results obtained by histochemical studies on the clamping-cortisone ulcer in rats are reported. The clamping-cortisone ulcer was made by the procedures mentioned before. In the histochemical studies, PAS, Alcian-blue, TBM. Aldehyde Fuchsin and neutral red staining methods were used for examining Polysaccharides changes in the tissue. Further, Gomori's and Azo staining methods were used for studing alkaline phosphatase. The animals were divided into two groups, i.e. the group treated by Clamping alone (the control group) and the group treated by the Clamping-Cortisone method. The PAS staining of the mucous content in the regenerative mucosa and the connective tissues of the base of the ulcer developed earlier and was stronger in the control group compared with that of the Clamping-Cortisone group. At the base of the ulcer, a clear difference of the staining in the middle and base layer was observed between the control group and the Clamping-Cortisone group. The degree of staining with PAS in the mucous membrane in the non-ulcerous region was also stronger in the control group compared with that in the Clamping-Cortisone group. In the Alcian-blue staining, the same results were obtained, but Alcian-blue staining activity became lower in the latter stages of the ulcer. The fibroblast and Collagen fiber were not stained. The spaces between the Collagen fibers were stained strongly. The TBM staining (pH2.5) degree did not show any remarkable difference between the control group and the Clamping-Cortisone group. Staining activity of the Alkaline phosphatase in the regenerative mucosal cells and connective tissue in the base of the ulcer developed earlier and stronger in the control group compared with that of the C-C ulcer. From these findings one of the reasons of prolongation of the ulcer induced by the Clamping-Cortisone method seemed to be attributed to decrease of mucous secretion from the mucosal cells and of polysaccharides in the connective tissue. Furthermore, it is also classified that there is a close relationship between alkaline phosphatase activities and the healing of the ulcer.
It is well known that the urea cycle and the metabolic pathway of glutamic acid in liver are chiefly involved in detoxication of ammonium. Pathogenesis of hyperammoniaemia, however, has not been completely understood up to date, though overproduction of ammonium in the intestine, intra- or extrahepatic portosystemic shunt formations or impaired hepatic ammonium detoxication were considered to be important aetiologic factors. It is very interesting problem whether or not there is the adaptability on detoxication of ammonium in the damaged liver, in which continuous inflow of ammonium produced in the intestine exists. In order to investigate on this problem, the experimental hyperammoniaemia was provided by parenteral administration of ammonium chloride in control rats and the rats with damagedliver by carbon tetrachloride, and then the enzymes, including all members in urea cycle and two of them in the metabolic pathway of glutamic acid and also the intermediates produced in these metabolic pathways, were assayed. It was shown that the activities of arginase, ornithine transcarbamylase, arginine synthetase, glutamic dehydrogenase and glutamine synthetase were increased in control rats after the repeated parenteral administration of ammonium chloride, and plasma contents of citrulline and glutamic acid were remained at the levels prior to the treatments. Therefore, it was reasonable to presume that various enzymes of urea cycle and the pathway of glutamic acid metabolism would be increased through the induction under the condition of continuous overproduction of ammonium in vivo, and could compensate the increased requirement of ammonium detoxication. On the other hand, after administration of ammonium chloride, in the rats with damaged liver, the induction of the enzymes in these metabolic pathways above was not encountered, the activities of the enzymes were rather decreased, and plasma contents of citrulline and glutamic acid were markedly increased. These findings indicated that there were lowered metabolic activities on the ammonium detoxication in the damaged liver, and these insufficiencies was further enhanced by the overproduction of ammonium. Six isoenzymes of glutamic dehydrogenase isolated from mitochondria) fraction of rat liver were found, however, the specific isoenzymes related to ammonium doxication were not evidenced. From these results above mentioned, it was concluded that impaired activities of hepatic enzymes in urea cycle and the metabolic pathway of glutamic acid were an essential aetiologic factor accounting for the incidence of hyperammoniaemia.
It is well known that ammonium produced in vivo is mainly detoxicated in the liver. With regards to the ammonium detoxication, there are two important metabolic pathways, i.e. urea cycle and glutamic acid pathway. The author clarified in the first report that enzymatic activities in these pathways were decreased in the liver of rats treated with CCl4. From the above experimental results, it is reasonable to assume that similar metabolic disturbances could be occurred in patients with serious liver diseases. In order to investigate on this problem in clinical materials, 115 patients with hepatic disease and 21 control patients were selected. To avoid the false estimation of hepatic disability on ammonium detoxication, resulting from measurement of a portion of ammonium escaping from the metabolic process in liver thru intra or extrahepatic porto-systemic shunt formations, the hepatocellular ability of ammonium detoxication was evaluated by the activities of glutamic dehydrogenase, glutamine synthetase in liver specimens obtained by needle biopsies, and plasma contents of glutamic acid. The activities of hepatic glutamic dehydrogenase and glutamine synthetase were decreased in the patients with serious liver diseases. The findings were most prominently encountered in the patients with severe hepatitis in which hepatic cell necrosis was highly recognized, and in those with hepatic coma accompanied by hyperammoniaemia. All cases of liver cirrhosis with hyperammoniaemia showed low activities of hepatic glutamic acid dehydrogenase and glutamine synthetase with high plasma content of glutamic acid. It was noteworthy that the same results were obtained even in the cases of porto-systemic encephalooathy, in which hepatic cell damage was not so prominent. From these results above mentioned, it was concluded that the impaired activities of hepatic enzymes in urea cycle and metabolic pathway of glutamic acid were an essential aetiologic factor accounting for the incidence of hyperammoniaemia as well as intra- or extra hepatic porto-systemic shunt formations.
The presence of the reduction products of bilirubin and the so-called 380 mu-substance of Ostrow in C-bile obtained by a duodenal tube from 3 cases of Gilbert's syndrome treated with phenobaribital was studied. The results are briefly summarized as follows. 1. During the administration of phenobarbital mesobilirubin was detected in the C-bile of all the three cases and dihydromesobilirubin in one of them. 2. Likewise during the treatment the so-called 380 mu-substance of Ostrow was observed markedly in the C-bile. The 1-3 spots of the paper chromatograohy proved to be negative to the pentdyopent test, but the 4-7 spots were positive. The absorption maxium of the 5-7 spots after the diazo reaction was at 540-530 mu, and the absorption maximum of azo dye of the spot 4 was around 520 mu which coincided with that of azo dye of Gunn rat. 3. Among the important factors that induce the decrease in the serum Bilirubin level the findings stated in the preceding items seem to play an important role, in addition to the acceleration of the synthesis of Bilirubin glucuronide.