A rapid simple method of electrophoretic separation and quantitative measurement of human serum amylase isozymes was established, and applied to the differential diagnosis of hyperamylasemia. After electrophoretic separation of serum amylase isozymes on cellulose acetate strip, the strip was stained directly on the Bluestarch-agar plate by the liberation of soluble highly coloured blue dye as a result of amylase activity. The stained strip was then put in a densitometor to determine the amount-ratio of amylase isozymes by integration. From the initial total amylase activity and this amount-ratio, the quantitative measurement of each amylase isozyme was made. The co+nstancy of the quantitative measurement by this method was demonstrated. In normal human serum, two isozymes of amylase were detected. One was identical with the isozyme in saliva and the homogenate of the salivary gland, while the other was compatible with the isozyme in the pure intraductal pancreatic juice and the homogenate of the pancreas. The normal range of the former was 35-113 Somogyi units/dl, and that of the latter was 27-83 units/dl. The abnormal elevation of the former was seen in cases of parotitis and hepato-biliary disorders with hyperamylasemia. The abnormal increase of the latter was detected in cases of pancreatic disorders with hyperamylasemia and after the examination of endoscopic pancreatography. This method was thought to be useful in the differential diagnosis of hyperamylasemia.
The determination of amylse activity was made by a chromogenic method using blue starch polymer (Pharmacia, Daiich Chem. Ltd). Animal experiment swere done using rats, Donryu strain, about 200gm in body weight. Agar electrophoresis was performed by the method of Harada and Kitamura (1971), using amylose as substrate. The tissue distribution of amylase activities of rats showed very high activities of the pancreas and the salivary glands, and lower activities of other tissues than amylase activity in serum. Serum amylase of rat was separated into two parts by agar electrophoresis. One part was demonstrated at gamma-globulin area, and the other part was located at alpha2-beta-globulin area. It was confirmed that anamylse isoenzyme at gamma-globulin area is a pancreatic origin, since increased amylase activities in sera of rats with experimentally induced acute pancreatitis and amylase activities of the pancreas homogenates of rats were only demonstrated at gammaglobulin area. On the contrary, amylase activities of the homogenates of salivary glands of rats were solely located at alpha2-beta-globulin area, indicating that anamylase isoenzyme at alpha2-beta-globulin area is a salivary origin. Estimation of amylase isoenzymes was possible densitometriccaly on electrophoretic zymograms. Serum amylases of rats and crystalline porcine pancreatic alpha-amylase (Sigma) showed one peak by gel filtration using Sephadex G-75, and Sephadex G-100, but the zymograms of these materials by agar electrophoresis demonstrated amylase isoenzymes cleary separated, suggesting amylase isoenzymes of rats' sera are not the subnits of alpha-amylase.
Amylase isoenzymes of 109 sera of patients were investigated by agar electrophoresis. The patients were consisted of acute parotitis (11 cases), the parotis tumor (6 cases), acute pancreatitis confirmed by laparotomy (one case), partial resection of the pancreas at the surgical operation of stomach cancer (2 cases) hyperamylasemia after abdominal surgery (8cases). Carcinoma of the pancreas (8 cases), chronic calcifying pancreatitis (3 cases), and miscellaneous diseases indicating hyperamylasemia (70 cases). In sera of one patient with acute pancreatitis and patients showing hyperamylasemia after partial resection of the pancreas, an amylase isoenzyme located at gamma-globulin area (isoamylase P) was mostly demonstrated. On the contrary, an amylase isoenzyme located at phi-gamma globulin area (isoamylase S) was seen in all sera of patients with acute parotitis. This finding is coincide with the zymograms observed in rats' sera as previously reported by the authors. Therefore, it is evident that isoamylase P and S are respectively a pancreatic and salivary origin. However, two other amylase isoenzymes differ from isoamylase P and S were sometimes seen in sera of a number of cases. One isoamylase was located at the middle of isoamylase P and S (isoamylase X), and the other isoamylase was seen nearer the cathode than isoamylase S (isoamylase Y). Isoamylase X was only found in sera of patients having severe acute damage of the pancreas, and subsequent zymograms showed the disappearance of isoamylase X at recovery state of acute damage, indicating that isoamylase X is a pancreatic origin and may be an index of the presence of severe acute damage of the pancreas. Isoamylase Y was frequently seen in sera of patients with acute parotitis, or severe acute damage of the pancreas, showing the multiple origins of isoamylase Y. In sera of the patients with advanced stage of carcinoma of the pancreas, and with calcifying pancreatitis, amylase activity in serum was low and isoamylase S was dominant on the zymograms. Gel filtration of sera of 10 patients showing zymograms of the presence of isoamylases P, S, X, and Y by Sephadex G-100 demonstrated only one peak of serum amylase supporting that isoamylases of patients sera are not the subnits of alpha- amylase.
27 patients who had been treated surgically for cancer of the pancreas and the biliary tract and for cholelithiasis with jaundice were investigated by means of electron microscope and light microscope to know changes of the liver with bile duct obstruction. 1) Hepatic parenchimal alterations varied from area to area within the hepatic lobule, but the central zone of the lobule often demonstrated severe ones. In the electron microscopy, bile capillary alterations were present prior to changes of hepatic parechymal cells. 2) In general, changes of the liver were severe with patients who had elevated icterus index and prolonged bile duct obstruction. 3) A specific hepatic cell degenerative change was seen in acute purulent cholecystitis with jaundice. 4) The mitochondria-desmosome complex was present in few patient with malignant bilary duct obstruction. This finding suggests the alteration of ion transportation in severe bile duct obstruction. 5) In the extrahepatic obstruction, it was demonstrated that bile pigments-like granules was excreted into intercellular space of hepaticcells rapidly then to Disse's space. 6) Some changes of the endoplasmic reticulum, polysome, lysosome, bile capillary and other organs were observed in advanced biliarytract disease with jaundice. 7) The present of increased mitochondria and polysome were often seen in cancer of the pancreas and biliary tract.
Serum leucine aminopeptidase (LAP) activities were determined in normal and in various kinds of diseases by means of Okuda-Fujii's method, which consisted in measurement of ammonia liberated by the action of LAP with direct colorimetric determination of ammonia, and were compared with those determined by the method in which leucyl-β-naphthylamide as used as the substrate to clarify the clinical meaning of the elevation of LAP activities determined by the both methods. LAP activity determined by the former method (LAP-NH3) was significantly higher than those determined by the latter method (LAP-Nap) in hepatocellular damages such as acute hepatitis, and LAP activity determined by the latter method showed the tendency to be higher than those determined by the former in obstructive jaudice. It was demonstrated that the serum of normal rats and CCl4 treated rats contained several multi-forms of the enzymes (LAP-I and LAP-II) which showed different substrate specificities toward leucinamide and leucine-β-naphthylamide by the TEAE cellulose column chromatography. In normal rat, LAP-I activity was higher in the NH3-method, and LAP-II activity was higher in the Nap-method. Marked increase of LAP activity in CCl4 treated rats was considered to be caused by the marked increase of LAP-NH3 belonging to LAP-I, and slight increases of LAP-NH3 and LAP-Nap belonging to LAP-II.
Pancreozymin secretin test was carried out on 43 normal persons, 31 patients with chronic calcifying pancreatitis and 68 patients with choledocho- and chole-lithiasis. The diagnosis was established radiologically demonstrable calcification of the pancreas in chronic calcifying pancreatitis and all others were done at operation. Duodenal aspirates were collected for 90minutes after the stimulation. The collected duodenal fluid was divided into three 10-minute after pancreozymin administration (Biliary phase) and six 10-minute after secretin administration (Pancreatic phase). Besides classic judgement by three parameters, total volume, maximal bicarbonate concentration and total amylase output, changes of six parameters including Meulengracht value, volume, amylase concentration, amylase output, bicarbonate concentration and bicarbonate output, were observed at the interval of 10minute during 90minutes in order to detect a slight impairment of the exocrine pancreas. In normal persons, the bile flow ceased toward the end of the biliary phase and curves of other five parameters declined gradually toward the end of the pancreatic phase. In the patients with chronic pancreatitis, on the other hand, relatively plateau curves were observed all through the pancreatic phase. In the patients with the biliary diseases, reported as having a slight impairment of the exocrine pancreas by classic judgement these five parameters dropped quickely in the later parts of the pancreatic phase, differed clearlly from normal persons.