Nagoya Journal of Medical Science

Studies were made on the methods for urinary amylase determination, stability of the enzyme, isoamylase in urine, normal ranges, diagnostic value in various diseases (pancreatic and non-pancreatic), and relationship between urinary amylase excretion and other laboratory tests. The saccharogenic method of Somogyi was chosen after comparing the three methods of Somogyi, Wohlgemuth, and Van Loon. Somogyi method was modified and simplified to be used for a screening test of pancreatic diseases. Diagnostic values of urinary amylase were evaluated in 54 control subjects, in 73 patients with pancreatic disease, and in 51 with non- pancreatic disease using this simplified method. Urinary amylase excretion was found to be a more sensitive and reliable index than amylase concentration in serum or urine for the diagnosis of acute pancreatitis, also of chronic pancreatitis and of carcinoma. The amylase excretion well reflected the clinical course of pancreatitis and served as a good indicator of its convalescent care. Differential diagnosis may be facilitated by determining urinary amylase excretion in the diseases which have elevated amylase values.

Attempts have been made to clarify the effect of pyridoxine deficiency as well as its overdosis on growth of transplantable tumors in mouse and rat.
The growth of transplantable solid tumor in pyridoxine deficient animals was remarkably suppressed while that of ascites tumor was not. The tumor bearing host was administered with a large amounts of pyridoxine and pyridoxal phosphate resulting in no effect on either tumor growth or survival time of tumor bearing host. The incorporation of leucine into S-180 ascites tumor cells under pyridoxine deficiency was similar to that in pyridoxine added condition, in vitro. In contrast, the efflux of leucine from pyridoxine deficient tumor cells increasd in comparison with that of control tumor cells. Although there were no changes in ultra- structure of tumor cells, the concentration ability of amino acid and protein synthesis from amino acid were decreased in pyridoxine deficient cells.
In conclusion, the present investigation is further support for a role of pyridoxine in tumor growth.

Three groups of male albino rats were fed with synthetic diets-high fat, low glucose (high fat diet group), high protein, low glucose (high protein diet group), and normal diets (normal diet group)-for 400 days, and the influence of these diets upon carbohydrate metabolism were examined. The following results were obtained.
1) Rats in the high fat diet group increased greatest body weight among the three groups, 400 g on the 200th day, and thereafter, continued to be obese.
2) Glucose tolerance curves and insulin sensitivity tests demonstrated significantly impaired carbohydrate metabolism of rats in the high fat diet group.
3) The decrease of glucose utilization and the increase of gluconeogenesis were observed in the tested liver slices of rats in the high fat diet group.
4) Glucose utilization and insulin sensitivity of adipose tissues and diaphragms of rats in the high fat diet group were significantly reduced compared to other groups.
5) The addition of papain in the medium reduced the incorporation of glucose-U-C¹⁴ into CO₂ of diaphragms of rats in high fat diet group and normal diet group but in the case of adipose tissues, although increased incorporation of radioactivity was observed in normal diet group, decreased incorporation was apparent on high fat diet group suggesting the presence of some different membrane structure of adipose tissue in high fat diet group.
6) In the pancreases of rats in the high fat diet group, hypertrophy of Langerhan's island and fibrosis of the exocrine glands were observed at the same time with the high level of insulin content of the pancreases.
From the above results the author concluded that the disorder in the carbohydrate metabolism of rats in the high fat diet group resembles the metabolic disarrangement of maturity onset type of human diabetes mellitus. The phenomenon can be comprehensive as an adaptation to the increased oxidation of fatty acids and the metabolic changes due to the accumulated lipid at the adipose tissue, and it is also partly consistent with Randle's theory on glucose-fatty acid cycle. The results suggest that high rate of fat in diet can aggrevate carbohydrate metabolism in rat as well as in the human being.

Fatty acid peroxides have been found in the atherosclerotic human aorta. Glavind found a correlation between the extent of the atherosclerotic changes and the content of lipid peroxides. These lipid peroxides are very toxic to the tissues. There are several compounds in the tissues to counteract the toxicity of peroxides in vivo, i.e. SH compounds, ascorbic acid, or tocopherol.
· It may be inferred that an increase of peroxides and a decrease of SH compounds occur in the experimental atherosclerotic aorta and that the administration of such reducing substances as ascorbic acid or SH compounds reactivates the respiration of the aorta inhibited by peroxides and retards the atherogenesis. It may be also supposed that a decrease of SH compounds occurs in the human atherosclerotic aorta. From these considerations the present investigations were performed. In the rabbits fed cholesterol, a decrease of nonprotein SH content and an increase of TEA reactive substances were observed in the aorta at the stage when the reduction of respiratory activity was not revealed. Furthermore, a decrease of total SH content was observed· in the serum, while total SH, nonprotein SH contents and SDH activity were normal in the liver. In the rabbits fed cholesterol with concomitant administration of ascorbic acid or SH compounds, the grade of atherosclerosis and the abnormality of total cholesterol and total SH contents were improved. The development of atherosclerosis was retarded as compared with the control, while the abnormality of lipids in quantity and of fatty acid composition was not improved.
Nonprotein SH content in the human atherosclerotic abdominal aorta decreased as compared with the normal aorta.
From these results, it may be concluded on atherogenesis that a damage of protein induced by nonenzymatic peroxidizing lipid-protein reaction exists in the serum and aorta.

According to the free radical theory by Harman, free radicals formed under various conditiors can cause the changes in DNA, which may result in chromo- somal aberrations, and can initiate lipid peroxidation in subcellular and cellular membrane systems.
The accumulation cf lipofuscin with age .also supports the significance of lipid peroxidation on aging.
In this paper·;two lines of research were carried out.
In experiment 1, peroxidized methyl linoleate was administered to the rats with or without supplementation of antioxi.dant (α-tocopherol felurate) in order to observe the toxic effects and the possibility of absorption from gastrointestinal tract. The changes of total nitrogen, total lipid, phospholipid, and total tocopherol and TBA reactive substances, which were estimated by two different ways, were observed in the livers of rats. The changes in appearance and the decrease of body weight were recognized in peroxidized oil administered groups. Total lipid and total tocopherol decreased in the liver of peroxidized oil administered groups, whereas there was no significant difference in the rat'.o of total tocopherol per total lipid. TBA reactive substances by non-shaking method increased in per- oxidized oil administered groups. Considering the above findings, serious toxic effects due to the oral administration of peroxidized methyl linoleate could not be protected by supplementation of antioxidant, and the peroxidized methyl linoleate or the destroyed products of it might be absorbed directly from the gastrointestinal tract.
In experiment II, age trends of the same substances as estimated in experiment I and of water soluble antioxidants by DPPH method were investigated in the liver of rats and human subjects. In both, content of water-soluble antioxidants decreased significantly with age and in contrast, total tocopherol increased in mature adult and mature old. In the rat liver the increase of TBA reactive substances was associated with age. In the human liver TBA reactive substances by shaking method increased from the young to the mature adult and decreased again in the old senescense. Assuming from the results of TBA reactive sub- stances, the capacity of lipoperoxide formation reaches to maximum in mature adult and the amount of lipoperoxide formation may increase with age.

Since Glavind reported the existence of peroxides in the atheroma, many investigatiors have become concerned with the studies of peroxides in atherogenesis. But the investigation of this problem is not yet complete. There exist in the living body SH compounds and antioxidants which inhibit the production and in- crease of lipoperoxides.
In this paper, the changes of the antioxidant in the living body were investigated whether lipoperoxides have a role in atherogenesis or not. I investigated in rabbits the changes of SH compounds and ascorbic acid in the aorta, liver, and serum following cholesterol feeding and analysed the polarographic protein wave activity of the serum for the estimation of SH compounds. Topographical investigation on SH compounds and ascorbic acid were made with histochemical method on the atherosclerotic rabbit aorta. The changes of these compounds in the serum were investigated on the atherosclerotic patients.
In the aorta and serum of the rabbits, SH content was decreased after the cholesterol feeding. The content of ascorbic acid was not changed in the serum, but increased.in the aorta. The decrease in polarographic protein wave activity was observed in the serum. SDH activity decreased in the aorta. The decrease of SDH activity in the liver followed the decrease in the aorta. In the liver of the cholesterol-fed rabbit, no significant difference of SH and ascorbic acid content was observed compared with the control rabbit. Staining intensity oE SH compounds and SDH was decreased in the intima and the inner media, but that of ascorbicacid was increased. In the serum of the atherosclerotic patients, SH content tended to decrease. No significant difference of ascorbic acid content was found between the atherosclerotic ahd the nonsclerotic subjects.
It is assumed that in the course of atherosclerosis the denaturation of the proteins in the aorta and serum might be induced. This denaturation might be caused by nonenzymatic action of lipoperoxides.

Male albino (Wistar strain) rats were fed with ordinary laboratory chew, Oriental pellet, properly balanced with regard to protein, lipids, carbohydrates, minerals, and vitamins, and with free water supply for ten days. Thereafter, the rats were force-fed daily with 4.5 ml of 30% ethanol solution or glucose solution, isocaloric with the former, by a gastric tube for another two weeks. The respiratory control index and AMP/ 0 ratio, the parameters which represent the intactness of mitochondria and the efficiency of the oxidative phosphorylation of liver mitochondria, were measured with liver mitochondria of the control and the alcohol-fed rats. Both indexes were found to be significantly lowered in the alcohol-fed rats indicating that ethanol feeding causes partial uncoupling of the oxidative phosphorylation in the liver mitochondria. The addition of bovine serum albumin in vitro did not recover this partial uncoupling. In the alcohol-fed rats for t wo weeks, the concentration of NEFA, ethanol, and acetaldehyde in the serum were increased which suggests that the increase has close relation with the disturb- ance of mitochondrial functions in alcohol fed rats, as NEFA is one of the well-known uncouplers. In order to elucidate the effects of alcohol and acetaldehyde on the mitochondrial oxidative phosphorylation in vitro, different amounts of alcohol or acetaldehyde were added to the mitochondrial suspension of the control group. Addition of more than 40 µmoles of ethanoljml of medium or more than 10 ,umoles of acetaldehyde/ ml of medium impaired both the respiratory control index and AMP/O ratio.
It is noted that the minimum concentration of ethanol effective for the isolated mitochondrial respiratory control and AMP/ 0 ratio was 40 µmoles/ml, the concentration which could be observed in the serum of the alcohol fed rats in vivo, but that of acetaldehyde w~s too high to be observed in ethanol fed rats in vivo.