血清天门冬氨酸氨基转移酶线粒体同工酶检测在酒精性肝病中的应用价值

目的分析酒精性肝病患者检测天门冬氨酸氨基转移酶线粒体同工酶(m-AST)的临床价值。方法选取2013年3月至2015年3月在该院肝病科接受诊治的酒精性肝病患者61例(观察组),另选取同期体检健康者61例(对照组),检测其血清m-AST、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)及γ-谷氨酰转移酶(GGT)水平。结果与对照组比较,观察组血清m-AST、ALT、AST及GGT水平均升高,差异均有统计学意义(P0.05)。酒精性肝病患者中,脂肪肝、肝炎及肝硬化患者治疗后上述各指标差异均有统计学意义(P0.05)。结论血清m-AST可以同ALT、AST及GGT一起作为临床辅助诊断酒精性肝病的指标,具有一定的临床价值。     

血清天门冬氨酸氨基转移酶线粒体同工酶在酒精性肝病中的应用价值

目的探讨检测血清天门冬氨酸氨基转移酶线粒体同工酶(m-AST)在酒精性肝病中的临床应用价值。方法以该院在2015年1~12月期间收治的61例酒精性肝病患者(A组)和50例非酒精性肝病患者(B组)为研究对象,选取同期在该院进行体检的60例健康者为对照组,采集所有受检者的血清样品。采用酶联免疫吸附法测定受检者血清中m-AST、天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)和γ-谷氨酰转移酶(GGT)水平,并比较2组间的差异。结果 A组患者血清m-AST、AST、ALT和GGT水平明显高于B组和对照组,差异均有统计学意义(P0.05);给予治疗3个月后研究组中脂肪肝患者、肝炎患者及肝硬化患者血清m-AST、AST、ALT和GGT水平比较差异有统计学意义(P0.05);A组患者和B组患者m-AST检测的阳性率明显高于AST、ALT和GGT(P0.05)。结论临床上检测酒精性肝病患者血清中m-AST在内的血清标志物水平对疾病的治疗和监测具有一定指导意义。     

Proteinase K inactivation of cytosolic aspartate aminotransferase isoenzyme for measurement of human serum mitochondrial aspartate aminotransferase

We studied a new proteinase K assay method for human serum mitochondrial aspartate aminotransferase. We found that proteinase K showed no inactivation of human mitochondrial aspartate aminotransferase isoenzyme and complete inactivation of cytosolic aspartate aminotransferase. Previous studies have shown that selective proteolytic measurement for mitochondrial aspartate aminotransferase in serum using the protease 401 cleaved peptide bond at Leu 20 from the amino-terminal bond shows complete inactivation of cytosolic aspartate aminotransferase and slight inactivation of mitochondrial aspartate aminotransferase isoenzyme, depending on protease concentration. In this investigation, we found that the proteinase K method does not depend on protease concentration. The proteinase K enzyme inactivation of cytosolic aspartate aminotransferase is caused by the cleavage of the peptide bond at lieu 21 from the aminoterminal bond. In studies with various animal cytosolic aspartate aminotransferase isoenzymes, proteinase K almost completely inactivated cytosolic aspartate aminotransferase. Precision and correlation using proteinase K for measurement of serum mitochondrial aspartate aminotransferase in human showed a good coefficient of variation (within-run <4.45%) and a coefficient of correlation of r = 0.985 (N = 125). © 1993 Wiley-Liss, Inc.     

Lactate dehydrogenase isoenzyme patterns in serum of patients with metastatic liver disease

Total lactate dehydrogenase (LD, EC 1.1.1.27) and LD isoenzymes were determined in serum of 170 patients with metastatic liver disease, 35 of whom had multiple metastatic sites. Overall, values of LD were above normal for 78% of the 170 patients. Half of the patients had an isomorphic pattern of LD isoenzymes (i.e., relative increase in all five isoenzymes); the other half had an increased LD-4,5 pattern, mostly a solitary increase in LD-5 only. Of those patients with normal LD values, 92% had the increased LD-4,5 pattern, whereas 70% of patients with LD values greater than 350 U/L had an isomorphic pattern of LD isoenzymes. All 35 patients with multiple metastatic sites had LD activity greater than 350 U/L; in the majority of them (74%) it was greater than 500 U/L; in 31 (89%), the increase was isomorphic. The diagnostic efficiency of the combined LD greater than 225 U/L (upper limit of normal) and increased LD-4,5 test results was much better than that of LD greater than 225 U/L alone (93% vs 74%). We conclude that serum LD and LD isoenzymes should be determined in every patient with suspected liver metastatic disease. The isomorphic pattern of LD isoenzymes is apparently associated with higher values for total LD and was common among the patients with multiple metastatic sites.     

Lactate dehydrogenase isoenzyme pattern in sera of patients with malignant diseases

Total lactate dehydrogenase (LDH; EC 1.1.1.27) activity and the percentage distribution of LDH isoenzymes were determined in 127 patients with malignant diseases. A shift in the isoenzyme patterns was observed toward the M-type, with an increase in the percentage of LDH-4 and LDH-5 isoenzymes and a slight increase in total LDH activity of all patients. Serum samples from 68 of the patients contained an abnormal isoenzyme of LDH, "LDH-1 ex," that, on agarose gel electrophoresis at pH 8.6, migrated between albumin and LDH-1 isoenzyme. Chemotherapy, radiotherapy, or surgical removal of the tumor was accompanied by disappearance of this abnormal isoenzyme. The heat stability of LDH-1 ex isoenzyme appears to be similar to that of LDH-1 but greater than that of the other LDH isoenzymes. Statistical analysis of these data demonstrated a significant correlation between malignancy and the appearance of LDH-1 ex isoenzyme (P less than 0.001). In contrast, the relationship between LDH-1 ex isoenzyme and metastasis or anatomical location of the malignancy is not statistically important (P less than 0.1).     

Mitochondrial enzymes in human serum: comparative determinations of glutamate dehydrogenase and mitochondrial aspartate aminotransferase in healthy persons and patients with chronic liver diseases

We measured the activities of two mitochondrial enzymes, the mitochondrial form of aspartate aminotransferase (EC 2.6.1.1) and glutamate dehydrogenase (EC 1.4.1.2), in the serum of apparently healthy persons (n = 84) and patients suffering from chronic liver diseases (n = 43). The distribution of activities for glutamate dehydrogenase, but not mitochondrial aspartate aminotransferase, was sex-dependent. The upper limits of the reference intervals (99th percentile) at 37 degrees C were 3.2 U/L for mitochondrial aspartate aminotransferase, 6.4 U/L for glutamate dehydrogenase (women), and 11.0 U/L for glutamate dehydrogenase (men); there was a weak correlation between the activities of both mitochondrial enzymes (r = 0.439). In patients with chronic liver diseases we found a greater increase in the activity of glutamate dehydrogenase than of mitochondrial aspartate aminotransferase and the correlation between the two mitochondrial enzymes was stronger. The diagnostic sensitivity and specificity of either mitochondrial enzyme was less than that of total aspartate aminotransferase, alanine aminotransferase (EC 2.6.1.2), or gamma-glutamyltransferase (EC 2.3.2.2).     

Activities of mitochondrial aspartate aminotransferase and creatine kinase isoenzyme MB in serum following coronary bypass surgery

The mitochondrial isoenzyme of aspartate aminotransferase showed only slight increases in serum of twenty-seven patients after uncomplicated coronary bypass surgery, which contrasted the rapid and substantial increases in creatine kinase MB. In seven patients suffering perioperative infarction or serious complications, substantial increases in mitochondrial aspartate aminotransferase were detected and the elevations in creatine kinase MB were prolonged. Mitochondrial aspartate aminotransferase may appear as a specific marker of myocardial necrosis following coronary bypass surgery. The elevations of creatine kinase and creatine kinase MB were detected as early as 5 minutes after onset of coronary reperfusion and slightly higher activities were measured in coronary sinus blood than in systemic blood sampled simultaneously. Increases in mitochondrial aspartate aminotransferase, however, could first be measured 8 hours after reperfusion.     

Effect of chlorpromazine on transmigration of mitochondrial aspartate aminotransferase in rat liver

Cholestatic liver injury was experimentally induced in rats by administration of chlorpromazine (CPZ). The peak activity of mitochondrial aspartate aminotransferase (AST) released in serum was found to precede the peak of total AST activity. The liver mitochondria obtained from rats treated with CPZ was fractionated into two subfractions: one containing the intermembrane space, and the other containing the matrix and the membranes. As a results, the relative activity of AST in the intermembrane space was significantly increased at 12 h after CPZ administration. This result suggests that mitochondrial AST, which is dominantly located in the mitochondrial matrix, transmigrated to the intermembrane space via the inner membrane under the effect of CPZ administration.     

Determination of mitochondrial aspartate aminotransferase in serum

Two specific and sensitive immunoassay methods for the determination of mitochondrial aspartate aminotransferase (m-AST) are described. One is a sandwich enzyme immunoassay which measures immunologically active m-AST using polystyrene balls coated with anti-m-AST antibody and peroxidase-labelled anti-m-AST antibody as the second antibody. The detection limit of this assay was 10 micrograms/l. The other is a paper disk method which measures catalytically active enzyme bound to anti m-AST antibody-conjugate paper disks. The calibration curve was linear up to 250 U/l. These assay methods were used to monitor the level of m-AST in serum. From measurements obtained by both methods, the correlation between the concentration of m-AST protein and its activity was poor (liver diseases, r = 0.539; myocardial infarction, r = 0.774) confirming that an inactive form of m-AST exists in serum, and that the specific activity of serum m-AST differs in individual diseases.     

Mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in sera of patients after myocardial infarction

Mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase (AST) were studied in the sera of 42 patients following acute myocardial infarction and compared to creatine kinase (CK), lactate dehydrogenase (LDH) and alanine aminotransferase (ALT). Mitochondrial AST( ASTm ) was detected in 93% (39/42) of patients. Maximum recorded ASTm activity was 59.5 +/- 8.8 U/l and was found 39.4 +/- 3.5 hours after the onset of symptoms (chest pain) of myocardial infarction. In contrast the maximum recorded cytoplasmic AST ( ASTc ) activity was greater (327 +/- 23 U/l) and it occurred earlier (33.5 +/- 2.2 hours) after onset of infarction compared to ASTm . ASTm correlated significantly (p less than 0.05) with ASTc , LDH and ALT but not with total CK or CK-MB. ASTc correlated significantly (p less than 0.05) with total CK, CK-MB and LDH but not ALT. Maximum recorded ASTm activity was significantly associated with the clinical assessment of left ventricular failure ( Killip classification) but not with ventricular arrhythmias. In a subset of 15 patients evaluated with invasive hemodynamic measurements of cardiac output and pulmonary capillary wedge pressure. ASTm correlated significantly (p less than 0.05) and better than CK-MB with the hemodynamic assessment of left ventricular dysfunction. Thus ASTSm can be readily identified in sera of patients after acute myocardial infarction and may be of value in the evaluation of patients with acute myocardial infarction.     

Use of malate dehydrogenase immobilized on the dialyzer groove of the Autoanalyzer II for serum aspartate aminotransferase determination

Malate dehydrogenase (EC 1.1.1.37) was immobilized on the lower groove of the dialyzer plate used for serum aspartate aminotransferase determination in the AutoAnalyzer II system. Immobilization was effected by covalently attaching malate dehydrogenase to the inner surface of the groove which was previously activated by treatment with glutaraldehyde at room temperature. The immobilized malate dehydrogenase catalyzed the reaction between oxaloacetate and NADH to form NAD in the coupled reaction originally proposed by Karmen. Results of the present method correlated well with those obtained by the Technicon SMA II system in which malate dehydrogenase is in solution (n = 99; r = 0.99; t = 0.30). The activity of immobilized malate dehydrogenase on the dialyzer groove was sufficient to measure serum aspartate aminotransferase for at least one month with continuous use. The stability of immobilized malate dehydrogenase was also dependent on the number of samples determined. The dialyzer plate is a reusable solid matrix for malate dehydrogenase immobilization. The expense of the present method is only half the cost of the method in which malate dehydrogenase is in solution.     

Proteolytic measurement of mitochondrial aspartate aminotransferase in human serum

A new proteolytic measurement of serum mitochondrial aspartate aminotransferase was evaluated using cytosolic aspartate aminotransferase inactivating protease. Some of the proteases, such as, alpha-chymotrypsin, subtilisin and cytosolic aspartate aminotransferase inactivating protease 401 from Streptomyces species, also specifically inactivated cytosolic aspartate aminotransferase, but not mitochondrial, aspartate aminotransferase. The protease 401 was the most heat stable for storage and showed a higher inactivation rate for cytosolic aspartate aminotransferase--up to 7000 IU/L--more than 200-fold the upper limit. The coefficient of variation of the proteolytic method was less than 10%. Results by the present method correlated with those by the immunochemical method (r = 0.970) and the regression curve was Y = 0.95X + 1.60 (Y: immunochemical method; X: proteolytic method). In the present assay system, reference values for mitochondrial aspartate aminotransferase activity in 500 healthy people ranged from 2.0-7.2 U/L (mean 3.8 U/L).