Determination of human aspartate aminotransferase isoenzymes by their differential sensitivity to proteases

The effect of various proteases (trypsin, chymotrypsin, subtilisin, protease 401, and thermolysin) on the mitochondrial isoenzyme (m-AST) and cytoplasmic isoenzyme (c-AST) of human and swine aspartate aminotransferase (AST;EC 2.6.1.1) was evaluated. All procedures including the reaction with proteases and the subsequent determination of the AST activity were carried out in an automatic analyzer. The mammalian c-AST was efficiently inactivated by chymotrypsin, subtilisin and protease 401 while m-AST activity decreased very slowly with these proteases. Thermolysin and trypsin showed much less effect on c-AST activity. Especially, chymotrypsin at concentrations of 0.5-1.0 g/L inactivated human c-AST almost completely but showed no detectable inactivating effect on m-AST. Thus chymotrypsin appears to be the most suitable protease for the differential determination of AST isoenzymes in human serum. Further studies on the effects of proteases with AST from other species showed that Escherichia coli AST resembled mammalian m-AST while Pseudomonas AST resembled c-AST.     

Activity of serum aspartate aminotransferase isoenzymes in patients with acute myocardial infarction

Activities of aspartate aminotransferase (AST) isoenzymes were determined in serial serum samples from 40 cases of acute myocardial infarction, and compared with activities of creatine kinase, CK-MB isoenzyme, lactate dehydrogenase, and alpha-hydroxybutyrate dehydrogenase for temporal changes. Cytosolic (soluble) AST (s-AST) and mitochondrial AST (m-AST) respectively increased 6.6 and 9.0 h after onset of chest pain. The median time at which serum m-AST activity peaked (15.8 U/L, range 6.4-53.5 U/L) was 47.8 h after the onset of infarction, 19.8 h later than the peak s-AST activity (171 U/L, range 53-517 U/L) and m-AST also disappeared from the serum more slowly than s-AST (p less than 0.001). Serum m-AST values were above normal for at least six days after the infarct. The ratio of m-AST to total AST in serum increased after myocardial infarction, being greatest (20%, range 11-32%) on the third day after onset. For individuals, peak activities of s-AST correlated well with total CK (r = 0.91) and CK-MB (r = 0.86) peak activities, indicating that s-AST also reflects the infarct size. However, m-AST correlated poorly with the enzymes commonly used in infarct diagnosis; it apparently provides different biological information.     

Effects of therapeutic coronary reperfusion on aspartate aminotransferase isoenzymes in sera of patients with acute myocardial infarction

We examined the kinetics of the catalytic activities of aspartate aminotransferase (AST, EC 2.6.1.1) isoenzymes in serum of 28 patients with myocardial infarction who were to receive either intracoronary urokinase--reperfusion angiographically proved--or conventional therapy (control group). Cytosolic (soluble) AST (s-AST) activity in serum increased rapidly immediately after recanalization, reaching a maximum 12 h after the onset of infarction. In the control group, this peak was reached 28 h after the onset (P less than 0.001). Peak s-AST activity was similar in the two groups. Peak activity and peak time for mitochondrial AST (m-AST) were the same for the two groups of patients; intervention that affects myocardial perfusion caused only a slight additional increase in m-AST activity in the early post-infarct period. There may be advantages to measuring m-AST, which is briefly influenced by reperfusion, instead of the usual cytosolic enzymes for assessment of myocardial damage in patients with myocardial infarction treated with thrombolytic therapy.     

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临床价值

目的探讨血清天门冬氨酸氨基转移酶线粒体同工酶(m-AST)活性在酒精性肝病(ALD)预后判断中的价值。方法采用免疫抑制法检测104例ALD患者[ALD组,包括酒精性脂肪肝(AFL)36例、酒精性肝炎(AH)52例和酒精性肝硬化(AC)16例]和100例病毒性肝炎患者[非酒精性肝病(NALD)组]治疗前(入院时)、治疗3周后及100名健康成人(对照组)血清m-AST活性,同时测定血清γ-谷氨酰基转移酶(GGT)、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)活性;观察AFL、AH及AC患者治疗前、后GGT、ALT、AST、m-AST活性的变化。结果 ALD组、NALD组血清GGT、ALT、AST、m-AST活性均明显高于对照组(P<0.05),NALD组各项指标明显高于ALD组(P<0.05)。治疗后AFL、AH及AC组血清GGT、ALT、AST活性均明显下降(P<0.001),AFL和AH组下降幅度明显高于AC组;AFL组血清GGT、ALT、AST、m-AST活性基本降至正常水平;AH组血清m-AST活性有明显下降,但AC组下降不明显。结论血清m-AST对ALD的治疗监测具有一定的临床意义。     

酶抑制法测定m-AST试剂性能评价及其在肝病中的意义

目的评价酶抑制法测定天门冬氨酸氨基转移酶（AST）线粒体同工酶（m—AST）活性的性能,并探讨m—AST在肝脏疾病中的临床价值。方法采用酶抑制法检测m—AST活性[即采用蛋白酶完全抑制AST细胞质同工酶（c—AST）的活性,然后用速率法进行检测],并与免疫抑制法比较。采用酶抑制法检测259例肝病患者（包括急性肝炎43例、慢性病毒性肝炎95例、肝衰竭20例、重型肝炎11例、肝硬化代偿期40例、肝硬化失代偿期9例、原发性肝癌41例）及220名健康体检者（正常对照组）m—AST活性,并与AST比较。结果酶抑制法批内变异系数（CV）为0．59％～2．23％,批间CV为5．24％～6．23％;回收率为101．6％～108．0％,平均为104．97％;线性方程Y=0．997X一1．51,r=0．9999,m—AST活性在450U／L内线性良好;可完全抑制1500U／L的c—AST活性;与免疫抑制法结果呈高度正相关（r=0．9998）;溶血对m—AST检测结果干扰较大;以95％可信区间（孟±1．96s）初步确定m—AST参考区间男性为3．1～9．5U／L,女性为2．5～8．7U／L。肝病患者m—AST活性均高于正常对照组（P〈0．05）,并与AST呈正相关。正常对照组m—AST／AST比值为0．30±0．07,肝病患者m—AST／AST比值均低于正常对照组（P〈0．05）,但变化不如m—AST活性明显。结论酶抑制法测定m．AST活性自动化程度高,结果准确可靠,重复性好,线性范围宽。m—AST活性能反映肝细胞损伤的严重程度,对肝脏疾病的临床分类和预后具有重要价值。     

Determination of aspartate aminotransferase isoenzymes in hepatic diseases — preliminary findings

The study sought to establish a relationship between the AST isoenzyme levels in serum and degree of hepatic damage, by using a new and simple immunochemical method for the differential determination of the isoenzymes. Sixty-nine patients with various hepatic diseases were studied.During hepatic damage, cytoplasmic isoenzyme (s-AST) is found in greater quantities than mitochondria! isoenzyme (m-AST), but the m-AST level increases to a greater extent in acute liver diseases. However, m-AST in alcoholic hepatitis is higher than expected from the total AST (t-AST) values. The ratio of m-AST to t-AST seems to discriminate alcoholic hepatitis from other liver diseases.     

Aspartate aminotransferase isoenzymes

Aspartate aminotransferase (AST, EC 2.6.1.1) exists in human tissues as two distinct isoenzymes, one located in the cytoplasm (c-AST), and the other in mitochondria (m-AST). Striated muscle, myocardium, and liver tissues are the main sources of AST. A growing body of information suggests that determination of AST isoenzymes in human serum is useful in evaluating damage to some of these organs. In hepatic disease, the test is used to assess liver necrosis and for determining prognosis. It may also assist in identifying patients with active alcoholic liver disease. In patients with acute myocardial infarction, measurement of AST isoenzymes provides diagnostic information that differs from that obtained by determination of total creatine kinase and lactate dehydrogenase enzymes, and their isoenzymes.     

A homogeneous assay system of aspartate aminotransferase iso-enzymes using proteases and application for clinical evaluation of myocardial infarction.

We designed a rapid, homogeneous assay for human aspartate aminotransferase (AST) isoenzymes, by a selective proteolysis of soluble AST (s-AST), using chymotrypsin and protease 401. The linearity of mitochondrial (m-AST) was elongated up to 4000 U/l. m-AST values from the human liver, and determined by a homogeneous assay using protease 401 or chymotrypsin, were relative to those obtained using an immunoprecipitation method. In perioperative patients or those with an acute myocardial infarction, the peaks of s-AST and m-AST values were noted 13 h and at 57 h after ictus, respectively, whereas the peak of ratio between was seen 6 h after ictus. In the case of Budd-Chiari syndrome, the maximum levels of the two AST activities were evident 14 days after hospitalization and the peak of ratio between them was seen after 7 days. We propose that this homogeneous assay can serve as a diagnostic tool for early phase detection of myocardial infarction and of Budd-Chiari syndrome.     

Enzyme immunoassay of human cytosolic aspartate aminotransferase

A sensitive and specific sandwich enzyme immunoassay (EIA) for human cytosolic aspartate aminotransferase (c-AST) has been developed. Serum was incubated with anti-c-AST antibody-coated polystyrene beads, and further incubated with anti-c-AST antibody-peroxidase conjugate. The peroxidase activity bound to the polystyrene bead was proportional to the amount of c-AST. The method allows measurement of serum c-AST ranging from 50-2,000 micrograms/l. No cross-reactivity with m-AST or other serum components was observed. Recovery, within-day precision, and day-to-day precision were good. The levels of c-AST obtained by the proposed EIA were compared with those based on enzyme activity. The results suggest that there is a considerable excess of immunologically active but catalytically inactive c-AST in normal and patient's sera, and that variable specific activities of c-AST are may be found in sera from different individuals.     

Optimal conditions for protease use in the assay of serum mitochondrial aspartate aminotransferase

The optimal conditions for selective proteolytic inactivation of cytosolic aspartate aminotransferase (c-AST) to determine mitochondrial aspartate aminotransferase (m-AST) in serum were studied. Protease 401 was found to be effective over a pH range of 6.0-10.0. A pH of 9.5 with 0.5% albumin in the reagent mixture was determined to be optimal for inactivation of c-AST and preservation of m-AST, lactic dehydrogenase (LDH), and malic dehydrogenase (MDH) in the assay procedure. The presence of serum endogenous protein inhibitors such as alpha 1-antitrypsin and alpha 2-macroglobin did not inhibit protease 401.     

Relative stabilities of purified human mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in lyophilized materials.

Eight different pools of purified human mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase were prepared, to examine the effects of the following matrix variables: the matrix support material (bovine serum albumin and polyvinylpyrrolidone), endogenous pyridoxal concentration, and azide as an antimicrobial preservation. Storage temperatures of 25 and 37 degrees C were used as a rapid and convenient means of accelerating the degradation process. Activity of the enzyme was measured with and without pyridoxal in the reaction solution. We found that the mitochondrial isoenzyme was consistently more labile than the cytoplasmic isoenzyme under identical storage conditions. Both isoenzymes were more stable in matrixes containing bovine serum albumin than in those containing polyvinylpyrrolidone. No apparent difference in the stability of either isoenzyme was observed at matrix pyridoxal concentrations of 15 micromol/L and 150 micromol/L. Only the mitochondrial isoenzyme in matrixes containing bovine serum albumin and 15 micromol of pyridoxal per liter had increased activity (about 9%) when pyridoxal was added to the enzymatic reagent. The amount of activity in reconstituted specimens did not apparently change after 72 h at 4 degrees C.     

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

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.     

Column-chromatographic separation of isoenzymes of aspartate aminotransferase.

We describe a column-chromatographic method for separating the mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in human serum. Bed height of the ion exchanger, pH, and salt concentrations in the eluting buffers are shown to be variables affecting the separation of the isoenzymes. Under the optimized conditions selected for this study, a 30% increase in volume was observed in one fraction, associated with changing the salt concentration of the eluting buffer and attributed to a contraction of the DEAE-Sephadex A-50. Elution profiles (enzyme activity vs. fraction number) were examined with highly purified mitochondrial and cytoplasmic isoenzymes of human origin in bovine serum albumin and human serum. Recovery of the enzyme in the eluted fractions averaged 102% (SD, 2.0%) for specimens prepared from the purified isoenzymes and 104% (SD, 10.7%) for 38 human serum specimens. The separation technique showed linearity to catalytic concentrations in excess of 200 U/liter (reaction temperature 30 degrees C) for each isoenzyme. Additional information is presented regarding among-day precision and the effect of specimen dilution.     

Prognostic value of mitochondrial aspartate aminotransferase in acute myocardial infarction

In 112 prospectively selected patients suffering from acute myocardial infarction (AMI), the serum CK, CK-MB, LD, HBD, AST and m-AST were determined from the time of admission to hospital and every 12 hours for three days in succession. Sixteen of the enrolled patients died due to complications which arose within the first four days of hospitalization while the rest had a favourable outcome. All enzyme activities were determined at 37 degrees C using routine methods; m-AST was measured using an immunochemical method. The statistical analysis of the results demonstrated that 12 hours after admission, serum m-AST and m-AST/AST ratio were significantly higher in the group of non-survivors compared with patients with a favourable prognosis. No significant differences in CK-MB were observed between survivors and non-survivors during the entire period. True and false positive rates were calculated for these and the other enzymes. An optimum decision level of 34 IU/L was chosen for m-AST and 10% for the m-AST/AST ratio. This gave a percentage of correctly classified patients, after 12 and 24 hours, of 74.9% and 91.9%, respectively. In conclusion, the immunochemical determination of m-AST in patients with AMI seems to be an early prognostic index which is able to distinguish patients with unfavourable outcome.     

Complexes of immunoglobulins A and G with aspartate aminotransferase isoenzymes in serum

We report the presence of complexes between aspartate aminotransferase (AST, EC 2.6.1.1) and immunoglobulin (Ig) in the serum of a patient suffering from lung cancer with metastasis to the liver. After fractionation of the serum by gel filtration, AST-Ig complexes (AST-IgA, AST-IgG) were demonstrated by counterimmunoelectrophoresis. Dissociating the complexes and recombining them with purified isoenzyme fractions, s-AST (cytoplasmic) and m-AST (mitochondrial), revealed that only s-AST binds to IgG, whereas IgA binds to both s-AST and m-AST. Although the association of AST with IgG has been reported, to our knowledge this is the first finding of both AST-IgA and AST-IgG complexes in a patient's serum. Serum AST-IgG complexes have been demonstrated in both healthy and diseased individuals; in the latter category, as reported here and by others, the liver is implicated.     

精浆天门冬氨酸氨基转移酶及其线粒体同工酶检测与精子膜功能完整性的关系

目的对精浆天门冬氨酸氨基转移酶(AST)及其线粒体同工酶(m-AST)的活性进行检测,并分析AST和m-AST活性与精子膜功能完整性的相关性。方法 122例精液标本,分别检测精浆AST和m-AST活性,用低渗肿胀-伊红Y结合试验(HOS-EY)检测精子头-尾膜完整性,用琥珀酸脱氢酶(SDH)染色法检测精子线粒体SDH,同时分析AST和m-AST活性与精子膜功能完整性的相关性。结果精浆AST与m-AST的活性分别为(238±125)U/L和(147±79)U/L,两者呈明显正相关性(r=0.740,P=0.000)。AST和m-AST活性均与(Ⅰ+Ⅱ+Ⅲ)型精子呈明显正相关性(r=0.443,P=0.013;r=0.691,P=0.000)。AST和m-AST活性均与Ⅳ型精子呈明显负相关性(r=-0.439,P=0.013;r=-0.689,P=0.000)。AST活性与精子SDH阳性率无明显相关性(r=-0.187,P=0.313),而m-AST活性与精子SDH阳性率呈明显负相关性(r=-0.471,P=0.007)。结论精浆AST和m-AST活性与精子膜功能完整性具有相关性,而m-AST活性检测更适合评价精子膜受损程度以及精子线粒体受损状况。     

Aspartate aminotransferase activity and isoenzyme proportions in human liver tissues.

Aspartate aminotransferase (EC 2.6.1.1) activity and the distribution of its isoenzymes in human liver were examined. Rabbit antiserum against porcin soluble (i.e., non-mitochondrial) enzyme cross-reacted with the soluble enzyme of human origin and was used in an immunoprecipitation assay to quantitate the soluble and mitochondrial isoenzymes. These were separated by rapid, semiquantitative electrophoresis on cellulose acetate and by three other quantitative techniques: isoelectric focusing and anion-and cation-exchange chromatography. The mitochrondrial enzyme averaged 81% of the total activity in normal adult human liver (n = 4). Its contribution was dramatically reduced in single specimens of human fetal liver (56% of total activity) and hepatoblastoma tissue (38%). Total enzyme activities (mumol min-1 per gram of tissue) were: adult, 150; fetal, 38; tumor, 6. Total enzyme concentrations (micromoles of enzyme per kilogram of tissue) found were: adult, 10.8; fetal, 2.7; tumor, 0.4. The concentrations and isoenzyme distribution in human liver are compared to those in various animal model systems. Other methods for quantitative estimation of the isoenzymes and their adaptability for use in estimating concentrations in serum are reviewed.     

40例酒精性脂肪肝患者血清酶指标检测分析

目的探讨酒精性脂肪肝(AFID)患者血清酶在病程中的变化规律及临床意义。方法对80例非酒精性脂肪肝(NAFID)、40例AFID、60例非脂肪肝(健康对照组)的血清酶等,进行测定分析。结果 AFID组血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、谷氨酰转肽酶(GGT)、天门冬氨酸氨基转移酶线粒体同工酶(mAST)、谷氨酸脱氢酶(GLDH)均显著高于健康对照组,差异有统计学意义(P0.01)。AFID组ALT、AST与NAFID组差异无统计学意义(P0.05),但AFID组的mAST、mAST/AST、GGT、GLDH均显著高于NAFID组,差异有统计学意义(P0.01)。结论 ALT、AST、GGT、mAST、GLDH活性测定有助于AFID的评估,有助于AFID的诊断及鉴别,具有重要的临床意义。     

Measurement of aspartate aminotransferase activity: effects of oxamate.

Oxamate, a potent inhibitor of lactate dehydrogenase, is shown also to inhibit aspartate aminotransferase activity, both in human serum and in purified isoenzymes of human origin. The inhibition was competitive with respect to 2-oxoglutarate for both isoenzymes. The apparent Ki was 29 mmol/L for the cytoplasmic enzyme and 17 mmol/L for the mitochondrial enzyme. Noncompetitive inhibition was found between oxamate and aspartate. At saturating concentrations of substrate (2-oxoglutarate greater than or equal to 15 mmol/L, L-aspartate greater than or equal 150 mmol/L) oxamate inhibited the mitochondrial enzyme but had less effect on the cytoplasmic isoenzyme. Oxamate at 40 mmol/L inhibited the enzyme in serum by 11 and 9% in assays containing 2-oxoglutarate at 6.7 and 15 mmol/L, respectively. This concentration of oxamate inhibited enzyme activity in serum by 5% more than did the same concentration of Cl- (itself an inhibitor). Oxamate (less than or equal to 30 mmol/L) had no measurable effect on the stability or activity of porcine malate dehydrogenase. Until the effects of its inhibitory properties are considered, addition of oxamate to suppress lactate dehydrogenase-mediated side reactions in the assay of aspartate aminotransferase cannot be recommended.     

Serum enzymes in acute myocardial infarction after intracoronary thrombolysis

Serum kinetics of total creatine kinase (CK), CK-MB isoenzyme, aspartate aminotransferase (AST), lactate dehydrogenase (LD) and alpha-hydroxybutyrate dehydrogenase (HBD) activities were studied in twenty patients with acute myocardial infarction randomly assigned to receive either intracoronary urokinase (group A) or conventional (control) therapy (group B). The temporal characteristics of enzyme changes described were the time lag from onset of chest pain until maximum catalytic concentration value, the rate at which enzymes are released into blood, the peak value of the serum enzyme curves and (d) the fractional disappearance rate (Kd) for each enzyme considered. Thrombolytic treatment induced earlier peak times in group A: for CK, 10.8 vs 27.0 h, for CK-MB, 10.4 vs 23.1, for AST, 13.9 vs 31.3, for LD, 24.4 vs 49.1, and for HBD, 20.5 vs 48.5 (for all enzymes, p less than 0.001). The maximal rate of release for the enzymes was at least twofold greater in group A. Enzyme peak activities and Kd were not significantly different between the groups. The most significant discrimination between the two groups was obtained with AST peak time (Hartz overlap index (Oi) = 0.11) and CK-MB peak time (Oi = 0.12).