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Microheterogeneity analysis of serum transferrin before and after therapies to hepatocellular carcinoma
lntematiorud
Hepatology International Hepatology Communications ELSEVIER
4 (1995)
COmmunications
190-194
Microheterogeneity analysis of serum transferrin before and after therapies to hepatocellular carcinoma Yasufumi Suzuki*, Yutaka Aoyagi, Akira Naitoh, Osamu Isokawa,. Hirotaka Igarashi, Masahiko Yanagi, Takeshi Suda, Shigeki Mori, Hitoshi Asakura The Third
Division,
Department of Internal 757, Asahimachi-Dori-I-Bancho,
Medicine,
Niigata Niigata
University 951, Japan
School
of Medicine,
Received 19 May 1995; revision received 11 September 1995; accepted 21 September 1995
AMJPCt
The reactivity of transferrin (Tf) with concanavalinA (Con A) wasexaminedby crossed immuno-afinity electrophoresis(CIAE) of the serafrom patientswith hepatocellularcarcinoma(HCC). SerumTf wasseparatedinto three species - Cl (Con A-nonreactive), C2 (weakly reactive) and C3 (-reactive) - designatedconsecutivelyfrom the anode by CIAE. Nineteenpairsof serumsamples wereevaluatedbeforeand 1month after therapiesfor HCC by transcatheterarterial embolizationand/or percutaneous ethanol injection, and surgical resection.The significantdecrease in the meanpercentageof Tf-Cl species wasobservedafter the abovetherapies,althoughtherewasno statisticaldifferencebetweenthe serumTf concentration beforeand after therapies.Thus, the presentstudy suggests that the measurement of thesespecies would be usefulasan index of severaltherapeuticeffectson HCC, especiallyin the caseof cY-fetoprotein(AFP)- and des-y-carboxy prothrombin (DCP)-non-producing HCC. Keywordr:
l
Transferrin; ConcanavahnA; Hepatocellularcarcinoma
Corresponding author,Tel.: +81 25 2236161 (Ext. 2525); Fax: +81 25 2232658.
0928-4346/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved 0928-4346(95)00244-D
SSDI
Y. Suzuki
et al. /International
Hepatology
Communications
4 (1995)
190-194
191
1. Introduction Human transferrin (Tf), one of the most important serum glycoproteins, is well known as an iron transporter [l] of molecular weight 80,000 [2] containing two asparagine glycosylation sites [2,3]. Higher values of serum transferrin are found in the initial phase of acute hepatitis, in chronic iron deficiency and during pregnancy, and Tf has been regarded as one of the acute phase proteins in chronic and malignant liver diseases [4]. On the other hand, Kushner et al. [5] and de Jong et al. [6] reported that Tf could be a negative acute phase protein model in which the effect of protein synthesis on N-glycosylation is dissociated from the effect of the acute phase response on glycosylation. Therefore, an association between the rate of Tf synthesis and the glycosylation pattern does not occur. Some earlier works revealed that alterations of carbohydrate structures appear in the serum Tf molecule in alcoholism and liver cirrhosis (LC) [7-91. Although the presence of similar alterations was also reported on Tf sugar chains in HCC [lo- 121, it was uncertain that the analysis of Tf-glycan structures could be useful as a serological examination tool for HCC. Recently, we have shown that the percentages of Con A-non-reactive and -weakly reactive species in total Tf in patients with HCC is significantly higher than that in LC regardless of serum concentration of this protein, and that the measurement of these species is useful for the diagnosis of HCC (in press). The present study aims to show that the measurement of altered glycosylation of serum Tf by CIAE with Con A would be useful for the evaluation of therapeutic effects on HCC. 2. Materials and methods 2.1. Patients and sera The diagnosis of patients with HCC was determined by liver function tests, several imaging modalities and histological examination. The treatments to HCC were transcatheter arterial embolization, percutaneous ethanol injection and surgical resection. Nineteen pairs of sera before and 1 month after therapy was administered to patients with HCC were subjected to the present analysis. All sera were stored at -20°C until analyzed. 2.2. Crossed immune-af$nity electrophoresis (CIAE) CIAE was performed in 1% agarose in 20mM barbital buffer (pH 8.6) containing 2 mg/ml of soluble Con A (C-2014, Sigma Chemical Company, St. Louis, MO. USA), as previously described [13]. Serum samples were adjusted to the concentration of 20 mg/dl of Tf with 20mM barbital buffer (pH 8.6) containing 200 mM (Ymethyl-D-glucopyranoside. The first dimensional electrophoresis was performed at 10 V/cm for 4.5h with cooling and the second one was carried out overnight with cooling at 2V/cm in 1% gel containing monospecific goat anti-human Tf (Cappel, Organ Teknika Corporation, West Chester, PA. USA).
192
Y. Suzuki
et al. / Znternatiomd
Hepatofogy
Communications
4 (1995)
190-194
2.3. Concentration of serum Tf Serum Tf concentration was determined using a single radial immunodiffusion plate (Hoechst Japan Corporation, Tokyo, Japan). 2.4. Statistical analysis Statistical analysis was performed using paired t-test. Data are expressed as mean f S.D. (S.D.). 3. Results
Serum Tf was resolved into three species by CIAE in a gel containing Con A. Three species - non-reactive, weakly reactive and reactive with Con A - were designated consecutively from the anode as Cl, C2 and C3, respectively.
W) 16 1
n=l9 8.7k2.7
6.9f2.5
(pcO.009)
Before
After
Fig. 1. Comparison of percentages of Cl species of Tf using paired serum samples from 19 patients with HCC. Significant difference was observed between before and 1 month after therapies carried out to HCC (P < 0.009).
Y. Suzuki
et al. /International
Hepatology
Communications
4 (1995)
190-194
193
Nineteen paired serum samples were subjected to the present analysis before and after therapies to HCC, and the mean percentage of C 1 species significantly decreased after treatments (6.9 f 2.5%) as compared with that before (8.7 f 2.7%, P < 0.009, Fig. 1). The diminution of mean percentage of Cl and C2 species was also observed after therapies to HCC (31.4 f 5.2%) as compared with before (34.1 f 5.8%, P < 0.07). No significant difference was observed between serum concentration of Tf before and after therapies to HCC. 4. Discussion There are several molecular variants of Tf in HCC. For example, Campion et al. reported the presence of fucosylated triantennary glycan, tetraantennary and pentaantennary glycans on the Tf molecule synthesized by hepatoma cell line [lo]. Yamashita et al. also determined more than ten types of carbohydrate structures of Tf, with different reactivity with lectins, from HCC-bearing patients [ 111. However, clinical usefulness of measuring Tf species remains to be established. Con A binds specifically the biantennary carbohydrate chain with or without a fucose residue at the innermost N-acetylglucosamine residue. This binding ability of the carbohydrate chain disappears if the biantennary structures undergo modilication such as bisecting-glucosaminylation and further branching leading to the formation of tri- and tetra-antennary structures [14-161. On the other hand, N-acetylglucosaminyl-transferase III, IV and V catalyze the addition of N-acetylgucosamine through a P-linkage to the mannose of the trimannosyl core structure of N-linked oligosaccharides of glycoproteins [17]. These enzymes convert a Con A-reactive glycan into a Con A-non-reactive glycan with N-acetylglucosaminylation. Actually, increased activities of N-acetylglucosaminyltransferase III were reported in sera and liver tissues from patients with HCC and with LC as compared with those in healthy subjects [18]. Therefore, highly branched complex-type glycans are the candidates for the variations of carbohydrate chains of Tf in this study, and the activation of these enzymes are speculated. Although the precise molecular basis for these variations is not determined at present, the evidence shown in our previous work together with the present study clearly indicate not only that the measurements of Con Anonreactive (Cl) and-weakly reactive (C2) species of Tf are useful in the differential diagnosis between HCC and non-neoplastic liver disease (in press), but that the analysis of carbohydrate structures of Tf would be useful in the evaluation of therapeutic effects on HCC. The significant decrease of the mean percentage of Cl species of Tf was observed after several therapies for patients with HCC as compared with that before. However, the mean percentage of Cl species after therapies in the present study was higher than that of normal control (0.7 f 1.3%) in our previous work (in press). The reason for this phenomenon is that the Cl species of Tf in LC has already increased as compared with that in normal control. Needless to say, the percentage of Cl in HCC is significantly higher than that in LC and normal control. The degree of the increment of Cl species varied in each HCC, but an obvious decrease of Cl species was observed after effective treatment. Thus, we may say that carbohydrate-based
194
Y. Suzuki
et al. /Inrerna~ionaI
Heparology
Communications
4 (1995)
190-194
measurement of Tf species is a useful marker in monitoring patients with HCC before and after therapies, especially when positive reaction for neither a-fetoprotein nor des-y-carboxyprothrombin is observed. 5. References [1] Aisen P, Listowsky I. Iron transport and storage proteins. In: Snell EE, Boyer PD, Meister A, Richardson CC, eds. Annual review of biochemistry. Vol. 49. Palo Alto: Annual Reviews Inc., 1980; 357-93. [2] MacGiIliray RTA, Mendez E, Shwale JG, Sinha SK, Lineback-Zins J, Brew K. The primary structure of human serum transferrin. J Biol Chem 1983; 258: 3543-53. [3] Spik G, Bayard B, Foumet B, Strecker G, Bouquelet S, Montreuil J. Studies on glycococonjugate LXIV complete structure of two carbohydrate units of human serotransferrin. FEBS Lett 1975; 50: 296-9. [4] Meliconi R, Parracino A, Facchini A et al. Acute phase proteins in chronic and malignant liver diseases. Liver 1988; 8: 65-74. [5] Kushner I. The phenomenon of the acute phase response. Ann NY Acad Sci 1982; 389: 39-48. [6] de Jong G, Feelders RA, van Noort WL, van Eijk HG. Transferrin microheterogeneity as a probe in normal and disease states. Glycoconjugate J 1995; 12: 219-26. [7] Heegaard NHH, Hagerup M, Thomsen AC, Heegaard PMH. Concanavalin A crossed affinity immunoelectrophoresis and image analysis for semiquantitative evaluation of microheterogeneity profiles of human serum transferrin from alcoholics and normal individuals. Electrophoresis 1989; 10836-40. [8] Spik G, Debruyne V, Montreuil J. Alterations of the carbohydrate structure of human serotransfertin in liver diseases. In: Popper H, Reutter W, Kottgen E, Gudat F, eds. Structural carbohydrates in the liver. Base]: MTP Press Limited, 1983; 477-83. [9] Poupon RE, Papoz L, Sarmini H, Elinck R. A study of microheterogeneity of transferrin in cirrhotic patients. Clin Chim Acta 1985; 151: 245-51. [IO] Campion B, Leger D, Wieruszeski JM, Montreuil J, Spik G. Presence of fucosylated triantennary, tetraantennary and pentaantennary glycans in transferrin synthesized by the human hepatocarcinema cell line Hep G2. Eur J Biochem 1989; 184: 405-13. [ 11) Yamashita K, Koide N, Endo T, Iwaki Y, Kobata A. Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma. J Biol Chem 1989; 264: 2415-23. [12] Matsumoto K, Maeda Y, Kato S, Yuki H. Alteration of asparagine-linked glycosylation in serum transfertin of patients with hepatocellular carcinoma. Clin Chim Acta 1994; 224: l-8. [I31 Bog-Hansen TC. Crossed immune-affinoelectrophoresis, an analytical method to predict the results of affinity chromatography. Anal Biochem 1973; 56: 480-8. [14] Marz L, Hatton MWC, Berry LR, Regoeczi E. The structural heterogeneity of the carbohydrate of desialylated human transferrin. Can J Biochem 1982; 60: 624-30. [15] Fu D, Van Halbeek H. N-glycosylation site mapping of human serotransferrin by serial lectin allinity chromatography, fast atom bombardment-mass spectrometry, and ‘H nuclear magnetic resonance spectroscopy. Anal Biochem 1992; 206: 53-63. [I61 Baezinger JU, Fiete D. Structural determinations of concanavalin A specificity for oligosaccharides. J Biol Chem 1979; 254: 2400-7. [I71 Kobata A. Structural changes induced in the sugar chains of glycoproteins by malignant transformation of producing cells and their clinical application. Biochimie 1988; 70: 1575-85. [I81 Ishibashi K, Nishikawa A, Hayashi N et al. N-Acetylglucosaminyltransferase 111in human serum, and liver and hepatoma tissues: increased activity in liver cirrhosis and hepatoma patients. Clin Chim Acta 1989; 185: 325-32.
Hepatology International Hepatology Communications ELSEVIER
4 (1995)
COmmunications
190-194
Microheterogeneity analysis of serum transferrin before and after therapies to hepatocellular carcinoma Yasufumi Suzuki*, Yutaka Aoyagi, Akira Naitoh, Osamu Isokawa,. Hirotaka Igarashi, Masahiko Yanagi, Takeshi Suda, Shigeki Mori, Hitoshi Asakura The Third
Division,
Department of Internal 757, Asahimachi-Dori-I-Bancho,
Medicine,
Niigata Niigata
University 951, Japan
School
of Medicine,
Received 19 May 1995; revision received 11 September 1995; accepted 21 September 1995
AMJPCt
The reactivity of transferrin (Tf) with concanavalinA (Con A) wasexaminedby crossed immuno-afinity electrophoresis(CIAE) of the serafrom patientswith hepatocellularcarcinoma(HCC). SerumTf wasseparatedinto three species - Cl (Con A-nonreactive), C2 (weakly reactive) and C3 (-reactive) - designatedconsecutivelyfrom the anode by CIAE. Nineteenpairsof serumsamples wereevaluatedbeforeand 1month after therapiesfor HCC by transcatheterarterial embolizationand/or percutaneous ethanol injection, and surgical resection.The significantdecrease in the meanpercentageof Tf-Cl species wasobservedafter the abovetherapies,althoughtherewasno statisticaldifferencebetweenthe serumTf concentration beforeand after therapies.Thus, the presentstudy suggests that the measurement of thesespecies would be usefulasan index of severaltherapeuticeffectson HCC, especiallyin the caseof cY-fetoprotein(AFP)- and des-y-carboxy prothrombin (DCP)-non-producing HCC. Keywordr:
l
Transferrin; ConcanavahnA; Hepatocellularcarcinoma
Corresponding author,Tel.: +81 25 2236161 (Ext. 2525); Fax: +81 25 2232658.
0928-4346/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved 0928-4346(95)00244-D
SSDI
Y. Suzuki
et al. /International
Hepatology
Communications
4 (1995)
190-194
191
1. Introduction Human transferrin (Tf), one of the most important serum glycoproteins, is well known as an iron transporter [l] of molecular weight 80,000 [2] containing two asparagine glycosylation sites [2,3]. Higher values of serum transferrin are found in the initial phase of acute hepatitis, in chronic iron deficiency and during pregnancy, and Tf has been regarded as one of the acute phase proteins in chronic and malignant liver diseases [4]. On the other hand, Kushner et al. [5] and de Jong et al. [6] reported that Tf could be a negative acute phase protein model in which the effect of protein synthesis on N-glycosylation is dissociated from the effect of the acute phase response on glycosylation. Therefore, an association between the rate of Tf synthesis and the glycosylation pattern does not occur. Some earlier works revealed that alterations of carbohydrate structures appear in the serum Tf molecule in alcoholism and liver cirrhosis (LC) [7-91. Although the presence of similar alterations was also reported on Tf sugar chains in HCC [lo- 121, it was uncertain that the analysis of Tf-glycan structures could be useful as a serological examination tool for HCC. Recently, we have shown that the percentages of Con A-non-reactive and -weakly reactive species in total Tf in patients with HCC is significantly higher than that in LC regardless of serum concentration of this protein, and that the measurement of these species is useful for the diagnosis of HCC (in press). The present study aims to show that the measurement of altered glycosylation of serum Tf by CIAE with Con A would be useful for the evaluation of therapeutic effects on HCC. 2. Materials and methods 2.1. Patients and sera The diagnosis of patients with HCC was determined by liver function tests, several imaging modalities and histological examination. The treatments to HCC were transcatheter arterial embolization, percutaneous ethanol injection and surgical resection. Nineteen pairs of sera before and 1 month after therapy was administered to patients with HCC were subjected to the present analysis. All sera were stored at -20°C until analyzed. 2.2. Crossed immune-af$nity electrophoresis (CIAE) CIAE was performed in 1% agarose in 20mM barbital buffer (pH 8.6) containing 2 mg/ml of soluble Con A (C-2014, Sigma Chemical Company, St. Louis, MO. USA), as previously described [13]. Serum samples were adjusted to the concentration of 20 mg/dl of Tf with 20mM barbital buffer (pH 8.6) containing 200 mM (Ymethyl-D-glucopyranoside. The first dimensional electrophoresis was performed at 10 V/cm for 4.5h with cooling and the second one was carried out overnight with cooling at 2V/cm in 1% gel containing monospecific goat anti-human Tf (Cappel, Organ Teknika Corporation, West Chester, PA. USA).
192
Y. Suzuki
et al. / Znternatiomd
Hepatofogy
Communications
4 (1995)
190-194
2.3. Concentration of serum Tf Serum Tf concentration was determined using a single radial immunodiffusion plate (Hoechst Japan Corporation, Tokyo, Japan). 2.4. Statistical analysis Statistical analysis was performed using paired t-test. Data are expressed as mean f S.D. (S.D.). 3. Results
Serum Tf was resolved into three species by CIAE in a gel containing Con A. Three species - non-reactive, weakly reactive and reactive with Con A - were designated consecutively from the anode as Cl, C2 and C3, respectively.
W) 16 1
n=l9 8.7k2.7
6.9f2.5
(pcO.009)
Before
After
Fig. 1. Comparison of percentages of Cl species of Tf using paired serum samples from 19 patients with HCC. Significant difference was observed between before and 1 month after therapies carried out to HCC (P < 0.009).
Y. Suzuki
et al. /International
Hepatology
Communications
4 (1995)
190-194
193
Nineteen paired serum samples were subjected to the present analysis before and after therapies to HCC, and the mean percentage of C 1 species significantly decreased after treatments (6.9 f 2.5%) as compared with that before (8.7 f 2.7%, P < 0.009, Fig. 1). The diminution of mean percentage of Cl and C2 species was also observed after therapies to HCC (31.4 f 5.2%) as compared with before (34.1 f 5.8%, P < 0.07). No significant difference was observed between serum concentration of Tf before and after therapies to HCC. 4. Discussion There are several molecular variants of Tf in HCC. For example, Campion et al. reported the presence of fucosylated triantennary glycan, tetraantennary and pentaantennary glycans on the Tf molecule synthesized by hepatoma cell line [lo]. Yamashita et al. also determined more than ten types of carbohydrate structures of Tf, with different reactivity with lectins, from HCC-bearing patients [ 111. However, clinical usefulness of measuring Tf species remains to be established. Con A binds specifically the biantennary carbohydrate chain with or without a fucose residue at the innermost N-acetylglucosamine residue. This binding ability of the carbohydrate chain disappears if the biantennary structures undergo modilication such as bisecting-glucosaminylation and further branching leading to the formation of tri- and tetra-antennary structures [14-161. On the other hand, N-acetylglucosaminyl-transferase III, IV and V catalyze the addition of N-acetylgucosamine through a P-linkage to the mannose of the trimannosyl core structure of N-linked oligosaccharides of glycoproteins [17]. These enzymes convert a Con A-reactive glycan into a Con A-non-reactive glycan with N-acetylglucosaminylation. Actually, increased activities of N-acetylglucosaminyltransferase III were reported in sera and liver tissues from patients with HCC and with LC as compared with those in healthy subjects [18]. Therefore, highly branched complex-type glycans are the candidates for the variations of carbohydrate chains of Tf in this study, and the activation of these enzymes are speculated. Although the precise molecular basis for these variations is not determined at present, the evidence shown in our previous work together with the present study clearly indicate not only that the measurements of Con Anonreactive (Cl) and-weakly reactive (C2) species of Tf are useful in the differential diagnosis between HCC and non-neoplastic liver disease (in press), but that the analysis of carbohydrate structures of Tf would be useful in the evaluation of therapeutic effects on HCC. The significant decrease of the mean percentage of Cl species of Tf was observed after several therapies for patients with HCC as compared with that before. However, the mean percentage of Cl species after therapies in the present study was higher than that of normal control (0.7 f 1.3%) in our previous work (in press). The reason for this phenomenon is that the Cl species of Tf in LC has already increased as compared with that in normal control. Needless to say, the percentage of Cl in HCC is significantly higher than that in LC and normal control. The degree of the increment of Cl species varied in each HCC, but an obvious decrease of Cl species was observed after effective treatment. Thus, we may say that carbohydrate-based
194
Y. Suzuki
et al. /Inrerna~ionaI
Heparology
Communications
4 (1995)
190-194
measurement of Tf species is a useful marker in monitoring patients with HCC before and after therapies, especially when positive reaction for neither a-fetoprotein nor des-y-carboxyprothrombin is observed. 5. References [1] Aisen P, Listowsky I. Iron transport and storage proteins. In: Snell EE, Boyer PD, Meister A, Richardson CC, eds. Annual review of biochemistry. Vol. 49. Palo Alto: Annual Reviews Inc., 1980; 357-93. [2] MacGiIliray RTA, Mendez E, Shwale JG, Sinha SK, Lineback-Zins J, Brew K. The primary structure of human serum transferrin. J Biol Chem 1983; 258: 3543-53. [3] Spik G, Bayard B, Foumet B, Strecker G, Bouquelet S, Montreuil J. Studies on glycococonjugate LXIV complete structure of two carbohydrate units of human serotransferrin. FEBS Lett 1975; 50: 296-9. [4] Meliconi R, Parracino A, Facchini A et al. Acute phase proteins in chronic and malignant liver diseases. Liver 1988; 8: 65-74. [5] Kushner I. The phenomenon of the acute phase response. Ann NY Acad Sci 1982; 389: 39-48. [6] de Jong G, Feelders RA, van Noort WL, van Eijk HG. Transferrin microheterogeneity as a probe in normal and disease states. Glycoconjugate J 1995; 12: 219-26. [7] Heegaard NHH, Hagerup M, Thomsen AC, Heegaard PMH. Concanavalin A crossed affinity immunoelectrophoresis and image analysis for semiquantitative evaluation of microheterogeneity profiles of human serum transferrin from alcoholics and normal individuals. Electrophoresis 1989; 10836-40. [8] Spik G, Debruyne V, Montreuil J. Alterations of the carbohydrate structure of human serotransfertin in liver diseases. In: Popper H, Reutter W, Kottgen E, Gudat F, eds. Structural carbohydrates in the liver. Base]: MTP Press Limited, 1983; 477-83. [9] Poupon RE, Papoz L, Sarmini H, Elinck R. A study of microheterogeneity of transferrin in cirrhotic patients. Clin Chim Acta 1985; 151: 245-51. [IO] Campion B, Leger D, Wieruszeski JM, Montreuil J, Spik G. Presence of fucosylated triantennary, tetraantennary and pentaantennary glycans in transferrin synthesized by the human hepatocarcinema cell line Hep G2. Eur J Biochem 1989; 184: 405-13. [ 11) Yamashita K, Koide N, Endo T, Iwaki Y, Kobata A. Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma. J Biol Chem 1989; 264: 2415-23. [12] Matsumoto K, Maeda Y, Kato S, Yuki H. Alteration of asparagine-linked glycosylation in serum transfertin of patients with hepatocellular carcinoma. Clin Chim Acta 1994; 224: l-8. [I31 Bog-Hansen TC. Crossed immune-affinoelectrophoresis, an analytical method to predict the results of affinity chromatography. Anal Biochem 1973; 56: 480-8. [14] Marz L, Hatton MWC, Berry LR, Regoeczi E. The structural heterogeneity of the carbohydrate of desialylated human transferrin. Can J Biochem 1982; 60: 624-30. [15] Fu D, Van Halbeek H. N-glycosylation site mapping of human serotransferrin by serial lectin allinity chromatography, fast atom bombardment-mass spectrometry, and ‘H nuclear magnetic resonance spectroscopy. Anal Biochem 1992; 206: 53-63. [I61 Baezinger JU, Fiete D. Structural determinations of concanavalin A specificity for oligosaccharides. J Biol Chem 1979; 254: 2400-7. [I71 Kobata A. Structural changes induced in the sugar chains of glycoproteins by malignant transformation of producing cells and their clinical application. Biochimie 1988; 70: 1575-85. [I81 Ishibashi K, Nishikawa A, Hayashi N et al. N-Acetylglucosaminyltransferase 111in human serum, and liver and hepatoma tissues: increased activity in liver cirrhosis and hepatoma patients. Clin Chim Acta 1989; 185: 325-32.