IN SERA OF PATIENTS WITH HEPATOCELLULAR CARCINOMA*1. nique, as one of diagnostic indices for hepatoma.
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1 IN SERA OF PATIENTS WITH HEPATOCELLULAR CARCINOMA*1 Norio SAWABU, Masatoshi NAKAGEN, Masao YONEDA, Hiroshi MAKINO, Shoni KAMEDA, Kenichi KOBAYASHI, Nobu HATTORI, and Masaru ISHII*3 First Department of Internal Medicine, School of Medicine, Kanazawa University,*2 and Division of Serology, Saitama Cancer Center*3 was fractionated by using polyacrylamide gradient gel electrophoresis in sera from 224 patients with various hepatobiliary diseases, including 63 with hepa- Band II, which was seen in the region near the ceruloplasmin, was detected in 22 of 63 patients with hepatoma (35%), but not in patients with other diseases. In addition to the II band, an extra band (II'), which was present between II and III, was found in 13 of these 22 hepatoma patients. None of other bands was nique, as one of diagnostic indices for hepatoma. hepatocellular carcinoma. These observations the detection of hepatocellular carcinoma. However, it has been widely accepted that in about 20% of patients with hepatocellular carcinoma, the content of AFP is as low as 200ng/ml, which is commonly observed in patients with hepatic disease other than hepatocellular carcinoma. Experimental liver cancer studies indicated that hepatoma tissue acquired carcinoembryonic properties GTP activity is quite low in the adult liver whereas the activity is extremely high in the fetal liver and in the hepatocellular carcinoma,3.11,20) and its activity has been found to become higher in some of patients with might occur during hepatocarcinogenesis, by acquisition of fetal properties by hepatocellular carcinoma, and that this fetal isoenzyme would be detectable in patients with hepatocellular carcinoma. To test this, we tally by using the polyacrylamide gradient gel electrophoresis, and discovered the novel specific to patients with hepatocellular carcinoma. MATERIALS AND METHODS Sera were obtained from 224 patients with hepatocellular carcinoma, 24 with metastatic liver carcinoma, 29 with liver cirrhosis, 25 with *1 This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, and from the Ministry of Health and Welfare.
2 chronic hepatitis, 16 with chronic alcoholic liver injury, 5 with subacute hepatitis, 14 with acute hepatitis, 9 with intrahepatic cholestasis, 22 with cholelithiasis, 10 with carcinoma of the biliary tract, and 7 with pancreatic carcinoma. The diagnosis was retrospectively examined and was based on findings upon surgery, biopsy, autopsy, and clinical and laboratory examinations. In 42 out of 63 cases of hepatocellular carcinoma, the diagnosis was confirmed by autopsy or surgery, and established by AFP and/or selective celiac angiography, as well as other clinical and laboratory examinations in 21 cases. Electrophoresis was carried out with the apparatus Model GE-4 (Pharmacia Fine Chemicals, Uppsala) loaded with polyacrylamide gradient (from 4 to 30%) gel plates PAG 4/30 (Pharmacia Fine Chemicals) at a constant voltage of 350 V for 3.5 hr in 0.05M Tris-glycine buffer (ph 8.35). The electrophoresis was carried out in a cold room N. SAWABU, ET AL. After electrophoresis, the gel plate was incubated with the following substrate mixture for 2hr A and B: Sera from hepatocellular carcinoma. Bands II and II' are observed in A but not in B. scribed by Albert et al.1) Solution A (120ml) C: Serum from alcoholic liver injury. D: Serum and solution B (80ml) were mixed just before from obstructive jaundice due to choledocholithiasis. E: Normal serum protein stained with Amido Black. glutamyl-a-naphthylamide (Sigma Chemical Co., St. Louis, U. S. A.) in physiological saline. To dissolve the substrate, the suspension was kept in a boiling water bath for 5min. Solution B consisted of 50mM glycylglycine (Wako Chemical Ind., Osaka) and 2.5mM MgCl2 in 0.1M phosphate buffer (ph 7.4). Isoenzyme was stained by incubating with 100mg of Fast Garnet GBC (Sigma Chemical Co.) in 200ml of 0.1M phos- metric scans were made using Model 2400-S p-nitroanilide as a substrate and glycylglycine and AFP was determined by the radioimmunoassay Labs., Tokyo). RESULTS from hepatobiliary diseases, demonstrating various electrophoretic bands (A, B, C, D) and normal serum protein stained with Amido sera were separated into twelve bands, labeled I, II, II', III, IV, V, VI, VIIa, VIIb, VIIc, VIIIa, and VIIIb in decreasing order of mobility. The bands I, II, and VIIb were seen in the region near postalbumin, cerulo- and Villa corresponded to the location of protein stained with Amido Black, seen in the lowest column (E) in Fig. 1. The isoenzyme patterns in various disease states are summarized in Table I. The fastest moving bands, bands I and III, were observed in concentrated sera from normal subjects. In addition to these two bands, which can be seen in normal sera, bands IV, V, and VI appeared and the increased intensity in staining reaction of these bands was observed much more commonly in sera from pathological subjects. Additionally, extra bands Vlla, VIIb, VIIc, VIIIa, and VIIIb appeared with a little higher frequency in sera of patients with biliary obstruction and hepatic malignancy, but none of these bands was strictly characteristic for any group of patients studied. Gann
3 zyme in Sera of Patients with Various Diseases Fig. 2. Correlation between appearance of th e of AFP of patients with hepatocellular carcinoma
4 On the other hand, band II, which was seen in the region near ceruloplasmin, was detected in 22 of 63 patients with hepatocellular carcinoma (35%), but not in patients with other hepatobiliary diseases. In addition to band II, an extra band (II') which was present between bands II and III, was found in 13 of these 22 patients. Table II shows the results of examination for bands II and II' in sera of patients with various diseases. The incidence of II band was 35% in total cases with hepatocellular carcinoma, and corre- N. SAWABU, ET AL. with hepatocellular carcinoma. In view of GTP was more than 100mU/ml. Fig. 2 compares the serum levels of AFP in patients of hepatocellular carcinnoma with and without band II. Serum level of AFP was significantly higher in the cases with band II than in those without band II (P< 0.001), and the band II was not found in the cases whose level of AFP was lower than 270 ng/ml. DISCUSSION By histological and biochemical studies, a present in the liver of fetal and neonatal rats, but to decrease rapidly after birth so that the activity of this enzyme in adult rat liver may be approximately 1/30 to 1/100 lower than that in fetal rat liver.2,3) Experimental liver cancer studies showed the course of hepatocarcinogenesis by several hepatocarcinogens in mice and rats, and that it was significantly increased in the liver cells both during the precancerous stage and in the liver cell carcinomas.3,4,6,11,20) In addition, similarity in the kinetic and immunolog- dye-induced hepatoma and fetal rat liver was also observed by Taniguchi et al.19) These observations indicate that hepatoma tissue acquires carcinoembryonic properties with in our study is almost in agreement with the AFP. Clinically this enzyme activity of serum result by these authors except that by Warnock21) who showed the highest is markedly elevated not only during hepa- incidence toma but also in the metastatic carcinoma, irrespective of the presence or absence of obstructive jaundice. However, extremely in most tumor cells of hepatoma, while the activity is not noted in the tumor cells in metastatic tumor but only in bile canaliculi and sinusoidal wall adjacent to the tumor.18) These facts strongly suggest that the hepatoma cells, and that this fetal isoenzyme might be detectable in sera of patients this presumption we tried to examine clinically whether or not the hepatoma-specific patients with hepatoma. The band II and/or II' in the zymogram by means of our procedure were found only in, but not in all, the patients with hepatocellular carcinoma. That is, out of 63 patients, 22 exhibited band II and 13 did band II' in addition to band II, but patients with other hepatobiliary disease did not. From the results of isoenzyme patterns in various hepatobiliary diseases, we presumed that II and II' bands could be specific for hepatocellular carcinoma. To identify these bands as a hepatoma-specific from serum and hepatoma tissue obtained from patients with these bands, and studying their physicochemical, enzymic, and immunological properties, as well as their tissue origin. The phenomenon similar to that observed in our study has recently been reported in alkaline phosphatase (AP) isoenzyme by some authors8,14,16,21) who described the occurrence of a variant AP specific to hepatocellular carcinoma. They regarded this isoenzyme as one of carcinoembryonic proteins, specifically a carcinoplacental protein, although its exact tissue origin has not been established. Gann
5 (60%) on the basis of 5 cases tested. The REFERENCES variant AP in serum will have some diagnostic 1) Albert. S.. Orlowska, J., Orlowski, M., value in hepatocellular carcinoma. However, for a comparison of diagnostic value of novel further studies on a larger population of 3) Fiala. S., Fiala, A. E., Dixon, B., J. Natl. patients with hepatocellular carcinoma will be required. Some studies have been published on clini- in neoplasm of the liver, demonstrating that the predominant occurrence of slowly migrating fractions may be diagnostically helpful.7,9,12,13,15) Nevertheless, there has been no report except one by Fujisawa and colleagues5) on the existence of hepatoma- 8) Higashino, K., Hashinotsume, M., Kang, K-Y., Takahashi, Y., Yamamura, Y., Clin. hepatoma patient. They reported activity in globulin appears only in sera of hepatocellular carcinoma, though the incidence and clinical significance of this specific fraction were not shown. The isoenzyme bands which were method were II, II', and III. The former two bands can be detected only in the sera from hepatocellular carcinoma patients and not in sera of patients with diseases other than hepatocellular carcinoma, but band III can be seen not only in normal sera but also in pathological sera accompanied with increase of the intensity of staining reaction of this band. Thus, it may be important to distinguish these three bands by using the polyacrylamide gradient electrophoresis, and our results can indicate electrophoretic het- 20) Tateishi, N., Higashi, T., Nomura, T., (Received December 26, 1977) (1964). 2) Albert, Z., Rzuidlo, Z., Starzyk, H., Acta 4) Fiala, S., Fiala, E. S., J. Natl. Cancer Inst., 5) Fujisawa, K., Kurihara, N., Kojima, M., Takahashi, T. Tanaka, M., "Disease of (1976). S. Karger, Basel. 6) Harada, M., Okabe, K., Shibata, K., Masuda, H., Miyake, K. Enomoto, M., 7) Hetland, O., Andersson, T. R., Gerner, T., 9) Igartua, E. B., Domecq, R., Findor, J., 11) Kalengayi, M. M. R., Ronchi, G., Desmet, (1975). 12) Orlowski, M., Szczeklik, A., Clin. Chim. 13) Patel, S., O'gorman, P., Clin. Chim. Acta, 14) Portugal, M. L., Azevedo, M. S., Manso, 15) Staeffen, J., Ballan, P., Ferrer, J., Beylot, J., Series, C., Terme, R., Pathol. Biol., 23, 16) Suzuki, H., Iino, S., Endo, Y., Torii, M., Miki, K., Oda, T., Ann. N. Y. Acad. Sci., 17) Tamaoki, H., Minato, S., Takei, S., Fuji- 665 (1974). 19) Taniguchi, N., Saito, K. Takakuwa, E., Naruse, A. Nakashima, K., Shinozaki, H., 21) Warnock, M. R., Reisman, R., Clin. Chim.
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