Bengt W. Johansson: The isoenzymes most often discussed in the literature is CK-MB. But LD isoenzymes are of great help in patients hospitalized late after symptoms and in order to differentiate between an LD
elevation due to myocardial damage and hepatic leakage secondary to a cardiac decompensation. In addition, A S A T isoenzymes might be of value.
Accurate determination of serum ASAT isoenzymes Aarne Konttinen and Kaarina Ojala Kivela Municipal hospital, HELSINKI, FINLAND
Abstract An improved electrophoretic modification for measuring aspartate aminotransferase (ASAT) isoenzymes is presented. This method fulfils the clinical requirements for sensitivity and allows the detection of 1 U/1 mitochondria ASAT activity at 25OC. The procedure is relatively simple, requiring about one hour for a series of 8 determinations. Mitochondria1 ASAT activity was found in all patients suffering from acute myocardial infarction pathological activity was observed for several days longer than that of total serum ASAT enzyme. None of the 25 healthy people studied had mitochondrial ASAT in their serum.
Efforts have been made to use the activity of aspartate aminotransferase (ASAT) isoenzymes to confirm acute myocardial infarction. ASAT consists of two isoenzymes which, in contrast to other isoenzymes, are not specific to certain organs. One is located in the cytoplasm and the other within the mitochondria (1). The release of mitochondrial ASAT isoenzyme into the serum thus indicates the degree of cell damage caused by necrotic injury to the mitochondria1 membranes. Of the several procedures proposed for the separation of ASAT isoenzymes, only electrophoresis is applicable in the clinical laboratory.
The earliest electrophoretic method which made feasible the quantitation of the isoenzymes (4, 5) is too insensitive for routine use. O u r new modification fulfils the requirements for sensitivity (1 U/I at 25OC) and is simple enough for day-to-day use. T h e procelure takes one hour, and only routine reagents are needed. The proposed method is based on an electrophoretic run on commercial cellulose acetate membranes, and the fractions of ASAT are visualized b y the colour reacticn with Fast Violet B. The Beckman Microzone system with barbital buffer pH 8.6 and ionic strength 0.075 M is used. T h e stock agar solution is made by melting 2 g agar (Difco Agar Noble) in 50 ml water in a boiling bath. The solution is divided into 4 ml fractions. These are stable for 2 weeks at 4OC. T h e substrate buffer contains Tris (hydroxymethyl) aminomet!iane, 1.2 g; L-aspartic aced (Merck), 2.66 g; 2-oxoglutaric acid (Merck); 350 mg, Polyvinylpyrrolidone, 3 g and Na-EDTA, 460 mg, dissolved in redistilled water, p H adjusted t o 7.4, and made up to 100 ml with distilled water. This buffer keeps for one month at 4OC. The substrate gel is prepared by melting 4 ml of the stock agar solution in a boiling water bath, mixing i t with 4 ml of substrate buffer, and with 0.25 ml of pyridoxalphosphate solution (KABI in tablet form, containing 0.83 mghablet) made by dissolving ,
113
one tablet in 1 ml of water. After mixing, the solution is poured immediately into a Petri dish. Pyridoxalphosphate solution must be prepared immediately before use, whereas the substarte gel will keep for several days a t 4OC. A serum volume of 0.25-1.00 pl, depending on the ASAT activity, is applied to the cellulose acetate strips with a running time of 20 minutes at 250 V. The membrane is then layered on to the substrate gel and incubated at 37OC for 30 minutes. After incubaticn, the isoenzymes are coloured as follows: another strip, soaked in Fast Violet 13 (40 mg/l ml of HsO, prepared just before use) and blotted slightly, is layered on the membrane (which is not removed from the gel) and left for 10 minutes at 37OC. The membrane is fixed by immersing in 5 % acetic acid for about two minutes, rinsing in water, and drying in air. The scanning is made at 540 nm with a Helena Auto Scanner (Fig. 1). The mitochondrial ASAT isoenzyme was not detectable in the serum of 25 healthy people studied. This is useful result as it strongly suggests that the enzyme is present
60
A S A l n o n a l limit
L 2
3
4
5
6
Fig. 2 . Daily measurements o f total ASAT and mitochondrial (m-ASAT) and MB isoenzyme of creatine kinase (CK-MB) in a patient having had acute myocardial infarction 24 hours earlier.
only in pathological processes in the body. In conformity with these findings, mitochondrial ASAT isoenzyme has not been detectable in healthy subjects by our earlier method or by more complicated and refined techniques (reviewed by Wilkinson) (6). I Boyde's (2, 3) results, however, derived with u a semiquantitative method, suggest that minimum activities of mitochondrial ASAT might be present in normal serum. We have found serum mitochondrial ASAT isoenzyme to be a very sensitive indicator of acute myocardial infarction. According to our findings, it is a considerably more accurate indicator for the detection of myocardial damage than total A S A T activity. The augmentation of mitochondrial ASAT appears to take place a few hours later, but remains detectable for several days longer, than total serum ASAT activity F i g . 1 . Representative scanning o f ASAT iso(Fig. 2). Thus the determination of mitoenzymes of a patient with acute myocardial in- chondrial ASAT might be used to help confarction. Total serum ASAT 40 UII, cytoplamatic firm an infarct, for instance, if the heart(c-ASAT) 34 UIl and mitochondrial (c-ASAT) specific isoenzyme of creatine kinase (CK6 U//. MB) is suspected of having disappeared from
z
2
114
the serum. Despite the superior specificity of serum CK-MB isoenzyme in myocardial damage, its use is handicapped by excessively quick disappearance from the serum. The determination of mitochondrial ASAT might be particularly valuable, for instance, where a negative CK-MB finding is suspected of being due to delayed admission. Because, in addition to the myocardium, mitochondrial ASAT isoenzyme is also present, for instance, in the liver, leakage from here into te serum might take place if mitochondrial membranes of cells are damaged sufficiently. An important point in the diagnosis of myocardial infarct is whether liver congestion is superimposed on the infarct, and whether such congestion is severe enough to cause rupture of the d w b l e membranes of the mitochondria. According to our earlier method, modest congestion of the liver was not followed by increased mitochondrial ASAT activity in the serum (5). O u r initial observations with the present method have not helped to solve this question, although massive liver damage has been observed to lead to the appearance of mitochondrial ASAT iscenzyme in the serum. In studying highly icteric sera, an additional band is formed on the strip because conjucated bilirubin reacts with the diazonium salt. This band does not move from the application groove and is thus clearly separable from the ASAT isoenzymes. The eventual clinical value of ASAT isoenzyme
determinations is still to be evaluated in further studies, for which the present sensitive and simple method appears to offer fresh possibilities.
References 1 . Boyd, J. W.: T h e intracellular distribution, latency and electrophoretic mobility of L-glutamate-oxaloacetate transaminase from rat liver. Biochem. J. 81:434, 1961. 2. Boyde, T. R. C.: Detection and assay of mitochondrial aspartate aminotransferase in serum. Z. Klin. Chem. 6: 431, 1968.
3. Boyde, T. R. C.: Serum levels of the mitochondrial isoenzyme of aspartate aminotransferase in myocardial infarction and muscular dystrophy. Enzym. Biol. Clin. 9: 385, 1968.
4. Murros, J., Konttinen, A. & Somer, H.: An electrophoretic method for the quantitation of aspartate aminotransferase isoenzymes. Clin. Chim. Acta 41: 263, 1972.
5. Murros, J., Konttinen, A. & Somer, H.: Mitochondria1 aspartate aminotransferase in myocardial infarction. Clin. Chim. Acta 48: 241, 1973. 6. Wilkinson, J. H.: Chemistry of enzymes of diagnostic interest. In T e Principles and Practice of Diagnostic Enzymology (ed. J. H. Wilkinson pp. 88-89. Edward Arnold Publ. Ltd, London 1976.
Creatine kinase isoenzymes in the confirmation of acute myocardial infarction Aarne Konttinen Kivela Municipal hospital, HELSINKI, FINLAND
Abstract Serum MB isoenzyrne of creatine kinase is the most specific and sensitive indicator of myocardial infarction. Despite these virtues, its clinical use is handicapped by rapid nor-
malization after the onset of myocardial injury. In patients suffering brain accidents, the increase of serum CK-MB isoenzymes indicates simultaneous myocardial damage and means poor prognosis.
115
elevation due to myocardial damage and hepatic leakage secondary to a cardiac decompensation. In addition, A S A T isoenzymes might be of value.
Accurate determination of serum ASAT isoenzymes Aarne Konttinen and Kaarina Ojala Kivela Municipal hospital, HELSINKI, FINLAND
Abstract An improved electrophoretic modification for measuring aspartate aminotransferase (ASAT) isoenzymes is presented. This method fulfils the clinical requirements for sensitivity and allows the detection of 1 U/1 mitochondria ASAT activity at 25OC. The procedure is relatively simple, requiring about one hour for a series of 8 determinations. Mitochondria1 ASAT activity was found in all patients suffering from acute myocardial infarction pathological activity was observed for several days longer than that of total serum ASAT enzyme. None of the 25 healthy people studied had mitochondrial ASAT in their serum.
Efforts have been made to use the activity of aspartate aminotransferase (ASAT) isoenzymes to confirm acute myocardial infarction. ASAT consists of two isoenzymes which, in contrast to other isoenzymes, are not specific to certain organs. One is located in the cytoplasm and the other within the mitochondria (1). The release of mitochondrial ASAT isoenzyme into the serum thus indicates the degree of cell damage caused by necrotic injury to the mitochondria1 membranes. Of the several procedures proposed for the separation of ASAT isoenzymes, only electrophoresis is applicable in the clinical laboratory.
The earliest electrophoretic method which made feasible the quantitation of the isoenzymes (4, 5) is too insensitive for routine use. O u r new modification fulfils the requirements for sensitivity (1 U/I at 25OC) and is simple enough for day-to-day use. T h e procelure takes one hour, and only routine reagents are needed. The proposed method is based on an electrophoretic run on commercial cellulose acetate membranes, and the fractions of ASAT are visualized b y the colour reacticn with Fast Violet B. The Beckman Microzone system with barbital buffer pH 8.6 and ionic strength 0.075 M is used. T h e stock agar solution is made by melting 2 g agar (Difco Agar Noble) in 50 ml water in a boiling bath. The solution is divided into 4 ml fractions. These are stable for 2 weeks at 4OC. T h e substrate buffer contains Tris (hydroxymethyl) aminomet!iane, 1.2 g; L-aspartic aced (Merck), 2.66 g; 2-oxoglutaric acid (Merck); 350 mg, Polyvinylpyrrolidone, 3 g and Na-EDTA, 460 mg, dissolved in redistilled water, p H adjusted t o 7.4, and made up to 100 ml with distilled water. This buffer keeps for one month at 4OC. The substrate gel is prepared by melting 4 ml of the stock agar solution in a boiling water bath, mixing i t with 4 ml of substrate buffer, and with 0.25 ml of pyridoxalphosphate solution (KABI in tablet form, containing 0.83 mghablet) made by dissolving ,
113
one tablet in 1 ml of water. After mixing, the solution is poured immediately into a Petri dish. Pyridoxalphosphate solution must be prepared immediately before use, whereas the substarte gel will keep for several days a t 4OC. A serum volume of 0.25-1.00 pl, depending on the ASAT activity, is applied to the cellulose acetate strips with a running time of 20 minutes at 250 V. The membrane is then layered on to the substrate gel and incubated at 37OC for 30 minutes. After incubaticn, the isoenzymes are coloured as follows: another strip, soaked in Fast Violet 13 (40 mg/l ml of HsO, prepared just before use) and blotted slightly, is layered on the membrane (which is not removed from the gel) and left for 10 minutes at 37OC. The membrane is fixed by immersing in 5 % acetic acid for about two minutes, rinsing in water, and drying in air. The scanning is made at 540 nm with a Helena Auto Scanner (Fig. 1). The mitochondrial ASAT isoenzyme was not detectable in the serum of 25 healthy people studied. This is useful result as it strongly suggests that the enzyme is present
60
A S A l n o n a l limit
L 2
3
4
5
6
Fig. 2 . Daily measurements o f total ASAT and mitochondrial (m-ASAT) and MB isoenzyme of creatine kinase (CK-MB) in a patient having had acute myocardial infarction 24 hours earlier.
only in pathological processes in the body. In conformity with these findings, mitochondrial ASAT isoenzyme has not been detectable in healthy subjects by our earlier method or by more complicated and refined techniques (reviewed by Wilkinson) (6). I Boyde's (2, 3) results, however, derived with u a semiquantitative method, suggest that minimum activities of mitochondrial ASAT might be present in normal serum. We have found serum mitochondrial ASAT isoenzyme to be a very sensitive indicator of acute myocardial infarction. According to our findings, it is a considerably more accurate indicator for the detection of myocardial damage than total A S A T activity. The augmentation of mitochondrial ASAT appears to take place a few hours later, but remains detectable for several days longer, than total serum ASAT activity F i g . 1 . Representative scanning o f ASAT iso(Fig. 2). Thus the determination of mitoenzymes of a patient with acute myocardial in- chondrial ASAT might be used to help confarction. Total serum ASAT 40 UII, cytoplamatic firm an infarct, for instance, if the heart(c-ASAT) 34 UIl and mitochondrial (c-ASAT) specific isoenzyme of creatine kinase (CK6 U//. MB) is suspected of having disappeared from
z
2
114
the serum. Despite the superior specificity of serum CK-MB isoenzyme in myocardial damage, its use is handicapped by excessively quick disappearance from the serum. The determination of mitochondrial ASAT might be particularly valuable, for instance, where a negative CK-MB finding is suspected of being due to delayed admission. Because, in addition to the myocardium, mitochondrial ASAT isoenzyme is also present, for instance, in the liver, leakage from here into te serum might take place if mitochondrial membranes of cells are damaged sufficiently. An important point in the diagnosis of myocardial infarct is whether liver congestion is superimposed on the infarct, and whether such congestion is severe enough to cause rupture of the d w b l e membranes of the mitochondria. According to our earlier method, modest congestion of the liver was not followed by increased mitochondrial ASAT activity in the serum (5). O u r initial observations with the present method have not helped to solve this question, although massive liver damage has been observed to lead to the appearance of mitochondrial ASAT iscenzyme in the serum. In studying highly icteric sera, an additional band is formed on the strip because conjucated bilirubin reacts with the diazonium salt. This band does not move from the application groove and is thus clearly separable from the ASAT isoenzymes. The eventual clinical value of ASAT isoenzyme
determinations is still to be evaluated in further studies, for which the present sensitive and simple method appears to offer fresh possibilities.
References 1 . Boyd, J. W.: T h e intracellular distribution, latency and electrophoretic mobility of L-glutamate-oxaloacetate transaminase from rat liver. Biochem. J. 81:434, 1961. 2. Boyde, T. R. C.: Detection and assay of mitochondrial aspartate aminotransferase in serum. Z. Klin. Chem. 6: 431, 1968.
3. Boyde, T. R. C.: Serum levels of the mitochondrial isoenzyme of aspartate aminotransferase in myocardial infarction and muscular dystrophy. Enzym. Biol. Clin. 9: 385, 1968.
4. Murros, J., Konttinen, A. & Somer, H.: An electrophoretic method for the quantitation of aspartate aminotransferase isoenzymes. Clin. Chim. Acta 41: 263, 1972.
5. Murros, J., Konttinen, A. & Somer, H.: Mitochondria1 aspartate aminotransferase in myocardial infarction. Clin. Chim. Acta 48: 241, 1973. 6. Wilkinson, J. H.: Chemistry of enzymes of diagnostic interest. In T e Principles and Practice of Diagnostic Enzymology (ed. J. H. Wilkinson pp. 88-89. Edward Arnold Publ. Ltd, London 1976.
Creatine kinase isoenzymes in the confirmation of acute myocardial infarction Aarne Konttinen Kivela Municipal hospital, HELSINKI, FINLAND
Abstract Serum MB isoenzyrne of creatine kinase is the most specific and sensitive indicator of myocardial infarction. Despite these virtues, its clinical use is handicapped by rapid nor-
malization after the onset of myocardial injury. In patients suffering brain accidents, the increase of serum CK-MB isoenzymes indicates simultaneous myocardial damage and means poor prognosis.
115
