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).     

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.     

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.     

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.     

An immunochemical procedure for determination of creatine kinase 3(1) (serum-specific) isoform in human serum evaluated

Changes in the proportions of individual isoforms of creatine kinase (CK) in serum promptly reflect both myocardial infarction and coronary reperfusion. A new commercial kit has been introduced for measuring CK-3(1) isoform in serum (ISOFOR-MM, International Immunoassay Labs.). This is an immunochemical assay containing CK-3(1) specific monoclonal antibody, bound to magnetizable particles, used to immunoextract this isoform. The CK activity of the sample is measured before and after immunoextraction and the difference in the two values gives the measure of CK-3(1). Extraction of CK-3(1) was complete at less than or equal to 1200 U/L. Analysis of between-day imprecision gave CV between 2.9-7.9%. The method was not susceptible to interference by CK-3(2) and CK-3(3) isoforms, CK-2 isoenzyme, or mitochondrial CK. Reference interval for CK-3(1) (expressed as percent of total CK-3) was 42-69%. Correlation between percent CK-3(1) by isoform electrophoresis (x) and evaluated procedure (y) was y = 0.83x + 7.6, with r = 0.957 (n = 40). The ISOFOR-MM performed well enough in this evaluation to replace electrophoresis or isoelectric focusing for measurement of CK-3(1) isoform.     

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.     

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.     

Immunoelectrophoretic characterization of human mitochondrial aspartate aminotransferase purified by ion exchange and affinity chromatography

Human mitochondrial aspartate aminotransferase (m-ASAT) was prepared for use as an antigen for antibody production and as a standard, i.e. a high degree of purity was demanded. The purification was performed by three ion exchange chromatography steps followed by affinity chromatography on aspartate coupled gel. Four preparations gave specific activities of 230 to 300 U/mg at 37°C. The purity of m-ASAT was assessed by crossed immunoelectrophoresis, which showed that the final preparation did not contain contaminating proteins. Immunization with the purified m-ASAT gave a good antibody response.     

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.     

Apoenzyme of aspartate aminotransferase in serum in health and disease

Aspartate aminotransferase activity has been measured in the sera of normal subjects and in patients with various diseases both with and without addition of pyridoxal phosphate to the assay medium. Considerable quantities of apoaminotransferase were present in almost all samples. In normal subjects this potentially-active enzyme corresponds on average to about half the holoenzyme present before reactivation with pyridoxal phosphate. However, considerable variations between individuals were found in the amounts of apoaminotransferase present in serum, and also between groups of patients with various diseases.