Clin Biochem, Vol. 23, pp. 327-334, 1990
0009-9120/90 $3.00 + .00 Cop]flight © 1990 The Canadian Society of Clinical Chemists.
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Clinical Usefulness of Malate Dehydrogenase and its Mitochondrial Isoenzyme in Comparison with Aspartate Aminotransferase and its Mitochondrial Isoenzyme in Sera of Patients with Liver Disease MAKOTO KAWAP and SEIJIN HOSAKI 2 1The Third Department of Internal Medicine, and 2Department of Laboratory Medicine, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo 113, Japan The activities of serum malate dehydrogenase (MDH) and its mitochondrial isoenzyme (MDHm) were studied in sera of patients with liver disease. They proved to be more useful than those of aspartate aminotransferase (AST) and its mitochondrial isoenzyme for detection of hepatocellular carcinoma and acute circulatory failure, and for estimation of the severity of acute hepatitis. The N/T value measuring system, which is adaptable for autoanalysis and allows simultaneous determination of activities depending on NAD and thionicotinamide adenine dinucleotide (thio-NAD), yields both the total activity of MDH and the N/T value which was correlated significantly with MDHm/MDH (r = 0.748). Assay of MDH and its mitochondrial isoenzyme in association with the N/T value measuring system seems to be more useful and less time consuming for estimation of the severity of liver diseases than that of AST and its mitochondrial isoenzyme.
KEY WORDS: malate dehydrogenase; aspartate aminotransferase; mitochondrion; isoenzyme; NAD; liver disease; ischemia; necrosis; hepatoma; hepatitis. Introduction
M
alate dehydrogenase (MDH, EC 1.1.1.37) is one of the enzymes of the tricarboxylic acid cycle and the malate-aspartate cycle (1). Serum MDH activity is increased in patients with liver or heart diseases. MDH has two isoenzymes: cytosolic and mitochondrial (MDHc and MDHm); an increase of MDHm activity in serum indicates severe cellular injury (2-4). Aspartate aminotransferase (AST; EC 2.6.1.1) also has cytosolic and mitochondrial isoenzymes (ASTc and ASTm), whose clinical usefulness in disease diagnosis and estimation of disease severity has been well investigated, especially in acute hepatic damage (5-9), including acute hepatitis (10)
Correspondence: Makoto Kawai, The Third Department of Internal Medicine, Tokyo Medical and Dental University, 5-45, Yushima, 1-chome, Bunkyo-ku, Tokyo 113, Japan. Manuscript received June 23, 1989; revised January 11, 1990; accepted January 15, 1990. CLINICAL BIOCHEMISTRY, VOLUME 23, AUGUST 1990
and alcoholic liver disease (11,12). However, such investigations of MDH and its isoenzymes are rare, and their clinical usefulness has not yet been sufficiently clarified. In this study, the activities of total MDH and its mitochondrial isoenzyme (MDHm) were assayed and compared with the activities of total AST and its mitochondrial isoenzyme (ASTm) in sera of patients with liver disease. In addition, the N/T value measuring system (13) was applied for analysis of MDH. A kinetic method based on nicotinamide adenine dinucleotide (NAD) and thionicotinamide adenine dinucleotide (thio-NAD), it simultaneously determines total activity and gives information on dehydrogenase isoenzyme composition. Materials a n d m e t h o d s PATIENT SAMPLES
Serum samples for enzyme assays Serum samples for determination of the reference range of MDH were obtained from 20 normal controls. For analysis of serum activity of MDH, AST, and their mitochondrial isoenzyme in patients with liver diseases including acute hepatitis, hepatocellular carcinoma, and acute circulatory failure, serum samples were taken from 44 patients when the activities of serum transaminases had almost reached peak levels (25 were also used for measurement of the N/T value defined in the next subsection). Acute viral hepatitis was diagnosed from the history and laboratory data of 30 patients. Hepatocellular carcinoma was diagnosed in seven patients by ultrasonography, computed tomography, and ~-fetoprotein level. Unless stated otherwise, their disease was active, and associated with tissue necrosis. Four showed acute elevation of serum transaminase activities in the terminal stage and fresh necrosis in the hepato327
KAWAI AND HOSAKI
cellular carcinoma without necrosis of liver parenchyma at autopsy; transcatheter arterial embolization (TAE) of hepatocellular carcinoma was successful in the three remaining patients. Acute circulatory failure was diagnosed in seven patients from the clinical course; the patients had suffered from congestive heart failure or shock followed by an episode of elevated transaminase activity which reached a peak level of >2000 U/L within 24-48 h, with marked elevation of lactate dehydrogenase activity (14,15).
Serum samples for the N/T value measuring system One hundred forty-one serum samples taken from 92 patients with liver diseases were used to analyze the correlation between MDH activity and the (N + T) value defined in the next subsection. Out of these, all 57 serum samples with MDH activity of 35-300 U/L, taken from 39 patients, were chosen to analyze the correlation between MDHm/MDH and the N/T value, since the correlation was less satisfactory outside this range. The serum samples used also included samples taken other than at the time of peak serum transaminase activities and multiple samples from the same patient. Data from these samples were used to describe the relationship between MDH and the (N+T) value, and between MDHm/MDH and the N/T value. The patients for analysis of the (N+T) and (N/T) values included: acute hepatitis: 27 (N+T), 20 (N/T); hepatocellular carcinoma: five (N+T), five (N/T); acute circulatory failure: three (N+T), three (N/T); chronic hepatitis: 15 (N+T), three (N/T); liver cirrhosis: seven (N+T); hepatocellular carcinoma without active necrosis: nine (N+T), two (N/T); metastatic liver tumor: one (N+T); alcoholic liver disease: eight (N+T), one (N/T); obstructive jaundice: 10 (N+T), one (N/T); drug induced hepatitis: two (N+T), one (N/T); primary biliary cirrhosis: one (N+T); suspicion of acute circulatory failure: four (N+T), three (N/T). Patients' diseases were diagnosed from the clinical course, laboratory data, ultrasonography, and computed tomography. The last four (N+T), three (N/T) patients were believed to have acute circulatory failure but were not definitely diagnosed due to lack of laboratory data. METHODS
Reagents Diethanolamine (DEA), sodium L-malate, phenazine methosulfate, nitro-blue tetrazolium and NAD were obtained from Wako Pure Chemical Industries, Osaka, Japan. Thio-NAD was obtained from Sigma Chemical Company, St. Louis, MO, USA. Universal Electrophoresis Film Agarose, for separation of MDH isoenzymes, was obtained from Corning Medical, Palo Alto, CA, USA. GOT ISOZYME TEST, for separation of ASTm, was obtained from Nippon Chemiphar Co., Tokyo, Japan. 328
Assay of MDH activity MDH was assayed according to the method of Wong and Smith (16).
Isoenzyme analysis of MDH by agarose gel electrophoresis A 4-~L sample was applied to the agarose film, and electrophoresis was performed with veronal buffer (pH 8.6, 0.06 mol/L) at 90 V for 40 min. A solution containing 0.07 mol/L DEA, 2.51 mmol/L NAD, 0.15 mmol/L phenazine methosulfate, 0.50 mmol/L nitro-blue tetrazolium, and 50 mmol/L sodium malate (pH 9.2) was used to stain the film at 37 °C for 20 min after electrophoresis. After decolorization was performed for approximately 15 min, with an adequate volume of acetic acid-alcoholdistilled water (1:14:5 v/v), the film was washed with water, dried, and examined densitometrically with a Cliniscan (Helena Laboratories Co., Beaumont, Texas, USA), and the MDHm/MDH value (%) was obtained. M D H m activity was calculated from the MDHm/MDH value and the MDH activity.
Assay of total AST activity AST activity was assayed according to the GSCC (17), using a Hitachi 736 analyzer (Hitachi Co., Tokyo, Japan). The reference range for the assay is 9-33 U/L.
Assay of ASTm activity ASTm was separated with a DEAE Sephadex A-50 microcolumn, GOT ISOZYME TEST (18), and assayed with a COBAS BIO centrifugal analyzer (F. Hoffmann-La Roche & Co., Basel, Switzerland); the reference range is
0009-9120/90 $3.00 + .00 Cop]flight © 1990 The Canadian Society of Clinical Chemists.
Printed in Canada. All rights reserved.
Clinical Usefulness of Malate Dehydrogenase and its Mitochondrial Isoenzyme in Comparison with Aspartate Aminotransferase and its Mitochondrial Isoenzyme in Sera of Patients with Liver Disease MAKOTO KAWAP and SEIJIN HOSAKI 2 1The Third Department of Internal Medicine, and 2Department of Laboratory Medicine, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo 113, Japan The activities of serum malate dehydrogenase (MDH) and its mitochondrial isoenzyme (MDHm) were studied in sera of patients with liver disease. They proved to be more useful than those of aspartate aminotransferase (AST) and its mitochondrial isoenzyme for detection of hepatocellular carcinoma and acute circulatory failure, and for estimation of the severity of acute hepatitis. The N/T value measuring system, which is adaptable for autoanalysis and allows simultaneous determination of activities depending on NAD and thionicotinamide adenine dinucleotide (thio-NAD), yields both the total activity of MDH and the N/T value which was correlated significantly with MDHm/MDH (r = 0.748). Assay of MDH and its mitochondrial isoenzyme in association with the N/T value measuring system seems to be more useful and less time consuming for estimation of the severity of liver diseases than that of AST and its mitochondrial isoenzyme.
KEY WORDS: malate dehydrogenase; aspartate aminotransferase; mitochondrion; isoenzyme; NAD; liver disease; ischemia; necrosis; hepatoma; hepatitis. Introduction
M
alate dehydrogenase (MDH, EC 1.1.1.37) is one of the enzymes of the tricarboxylic acid cycle and the malate-aspartate cycle (1). Serum MDH activity is increased in patients with liver or heart diseases. MDH has two isoenzymes: cytosolic and mitochondrial (MDHc and MDHm); an increase of MDHm activity in serum indicates severe cellular injury (2-4). Aspartate aminotransferase (AST; EC 2.6.1.1) also has cytosolic and mitochondrial isoenzymes (ASTc and ASTm), whose clinical usefulness in disease diagnosis and estimation of disease severity has been well investigated, especially in acute hepatic damage (5-9), including acute hepatitis (10)
Correspondence: Makoto Kawai, The Third Department of Internal Medicine, Tokyo Medical and Dental University, 5-45, Yushima, 1-chome, Bunkyo-ku, Tokyo 113, Japan. Manuscript received June 23, 1989; revised January 11, 1990; accepted January 15, 1990. CLINICAL BIOCHEMISTRY, VOLUME 23, AUGUST 1990
and alcoholic liver disease (11,12). However, such investigations of MDH and its isoenzymes are rare, and their clinical usefulness has not yet been sufficiently clarified. In this study, the activities of total MDH and its mitochondrial isoenzyme (MDHm) were assayed and compared with the activities of total AST and its mitochondrial isoenzyme (ASTm) in sera of patients with liver disease. In addition, the N/T value measuring system (13) was applied for analysis of MDH. A kinetic method based on nicotinamide adenine dinucleotide (NAD) and thionicotinamide adenine dinucleotide (thio-NAD), it simultaneously determines total activity and gives information on dehydrogenase isoenzyme composition. Materials a n d m e t h o d s PATIENT SAMPLES
Serum samples for enzyme assays Serum samples for determination of the reference range of MDH were obtained from 20 normal controls. For analysis of serum activity of MDH, AST, and their mitochondrial isoenzyme in patients with liver diseases including acute hepatitis, hepatocellular carcinoma, and acute circulatory failure, serum samples were taken from 44 patients when the activities of serum transaminases had almost reached peak levels (25 were also used for measurement of the N/T value defined in the next subsection). Acute viral hepatitis was diagnosed from the history and laboratory data of 30 patients. Hepatocellular carcinoma was diagnosed in seven patients by ultrasonography, computed tomography, and ~-fetoprotein level. Unless stated otherwise, their disease was active, and associated with tissue necrosis. Four showed acute elevation of serum transaminase activities in the terminal stage and fresh necrosis in the hepato327
KAWAI AND HOSAKI
cellular carcinoma without necrosis of liver parenchyma at autopsy; transcatheter arterial embolization (TAE) of hepatocellular carcinoma was successful in the three remaining patients. Acute circulatory failure was diagnosed in seven patients from the clinical course; the patients had suffered from congestive heart failure or shock followed by an episode of elevated transaminase activity which reached a peak level of >2000 U/L within 24-48 h, with marked elevation of lactate dehydrogenase activity (14,15).
Serum samples for the N/T value measuring system One hundred forty-one serum samples taken from 92 patients with liver diseases were used to analyze the correlation between MDH activity and the (N + T) value defined in the next subsection. Out of these, all 57 serum samples with MDH activity of 35-300 U/L, taken from 39 patients, were chosen to analyze the correlation between MDHm/MDH and the N/T value, since the correlation was less satisfactory outside this range. The serum samples used also included samples taken other than at the time of peak serum transaminase activities and multiple samples from the same patient. Data from these samples were used to describe the relationship between MDH and the (N+T) value, and between MDHm/MDH and the N/T value. The patients for analysis of the (N+T) and (N/T) values included: acute hepatitis: 27 (N+T), 20 (N/T); hepatocellular carcinoma: five (N+T), five (N/T); acute circulatory failure: three (N+T), three (N/T); chronic hepatitis: 15 (N+T), three (N/T); liver cirrhosis: seven (N+T); hepatocellular carcinoma without active necrosis: nine (N+T), two (N/T); metastatic liver tumor: one (N+T); alcoholic liver disease: eight (N+T), one (N/T); obstructive jaundice: 10 (N+T), one (N/T); drug induced hepatitis: two (N+T), one (N/T); primary biliary cirrhosis: one (N+T); suspicion of acute circulatory failure: four (N+T), three (N/T). Patients' diseases were diagnosed from the clinical course, laboratory data, ultrasonography, and computed tomography. The last four (N+T), three (N/T) patients were believed to have acute circulatory failure but were not definitely diagnosed due to lack of laboratory data. METHODS
Reagents Diethanolamine (DEA), sodium L-malate, phenazine methosulfate, nitro-blue tetrazolium and NAD were obtained from Wako Pure Chemical Industries, Osaka, Japan. Thio-NAD was obtained from Sigma Chemical Company, St. Louis, MO, USA. Universal Electrophoresis Film Agarose, for separation of MDH isoenzymes, was obtained from Corning Medical, Palo Alto, CA, USA. GOT ISOZYME TEST, for separation of ASTm, was obtained from Nippon Chemiphar Co., Tokyo, Japan. 328
Assay of MDH activity MDH was assayed according to the method of Wong and Smith (16).
Isoenzyme analysis of MDH by agarose gel electrophoresis A 4-~L sample was applied to the agarose film, and electrophoresis was performed with veronal buffer (pH 8.6, 0.06 mol/L) at 90 V for 40 min. A solution containing 0.07 mol/L DEA, 2.51 mmol/L NAD, 0.15 mmol/L phenazine methosulfate, 0.50 mmol/L nitro-blue tetrazolium, and 50 mmol/L sodium malate (pH 9.2) was used to stain the film at 37 °C for 20 min after electrophoresis. After decolorization was performed for approximately 15 min, with an adequate volume of acetic acid-alcoholdistilled water (1:14:5 v/v), the film was washed with water, dried, and examined densitometrically with a Cliniscan (Helena Laboratories Co., Beaumont, Texas, USA), and the MDHm/MDH value (%) was obtained. M D H m activity was calculated from the MDHm/MDH value and the MDH activity.
Assay of total AST activity AST activity was assayed according to the GSCC (17), using a Hitachi 736 analyzer (Hitachi Co., Tokyo, Japan). The reference range for the assay is 9-33 U/L.
Assay of ASTm activity ASTm was separated with a DEAE Sephadex A-50 microcolumn, GOT ISOZYME TEST (18), and assayed with a COBAS BIO centrifugal analyzer (F. Hoffmann-La Roche & Co., Basel, Switzerland); the reference range is
