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Increased CK-BB concentrations in serum following cardiac surgery
29
Clinica Chimica Acta, 93 (1979) 29-33 0 Elsevier/North-Holland Biomedical Press
CCA 10103
INCREASED CK-BB CONCENTRATIONS CARDIAC SURGERY
MARK H. ZWEIG * and ANDRfi Clinical Pathology Department, MD 20014 (U.S.A.) (Received
October
20th,
IN SERUM FOLLOWING
C. VAN STEIRTEGHEM
**
Clinical Center, National Institutes of Health, Bethesda,
1978)
Summary We measured the BB isoenzyme of creatine kinase by a specific radioimmunoassay in the serum of 47 patients following cardiac surgery. A sharp increase in CK-BB occurred immediately after surgery, with rapid return to baseline by the fourth post-operative day. This data, along with other reports in the literature, suggest that CK-BB is released into the circulation following myocardial insult.
Introduction Serum creatine kinase (CK) activity has received much attention recently by those interested in the diagnosis and management of myocardial infarction. Most recently, serial measurements have been used in attempts to estimate infarct size and to assess efficacy in interventions designed to limit infarct extension and to reduce complications. Many claims have come forth regarding the specificity and sensitivity of the MB or “myocardial” isoenzyme of CK for the diagnosis of acute myocardial infarction. As a result, there is widespread use of electrophoretic and anion exchange column chromatographic methods for separating and measuring CK-MB enzymatic activity in serum. Because of various problems with these methods, immunochemical approaches such as immunoinhibition, immunoprecipitation and radioimmunoassay have been applied to the measurement of CK-MB [l-5]. None of the radioimmunoassays is specific for the MB isoenzyme. All claim to measure B subunit activity in both the MB and BB isoenzymes. The assumption is made that the BB isoenzyme is negligible or absent, in spite of numerous reports to the contrary [ 61. Furthermore, CK-MB is still widely considered specific for acute myocar* To whom correspondence should be addressed. ** Present address: Clinical Chemistry Service, Vrije Universiteit Brussel. Universitair Ziekenhuis SintPieter. Hoogstreat, B-1000 Brussels, Belgium.
30
dial infarction even though it is found to be increased in serum in a wide variety of clinical situations. We have developed a sensitive radioimmunoassay for the BB isoenzyme which appears specific, havong only 1% crossrea~tivity with CK-MB [6]. In the course of measuring CK-BB in the serum of unselected patients, we found 20-fold elevations of CK-BB in two patients who had undergone cardiac surgery. Because of this finding we looked more closely at patients having cardiac surgery and found that virtually all had elevated serum CK-BB concentrations immediately after surgery, persisting for several days. Methods Forty-seven patients undergoing cardiac surgery, usually with cardiopulmonary bypass, were followed. The surgical procedures included coronary artery bypass graft, valve replacement, septal defect repair, ventricular myectomy, etc. Where possible, serum obtained post-operatively on the day of surgery and daily thereafter was assayed. Baseline values were established for each patient by assaying preoperative serum or post-operative serum five or more days after surgery. The radioimmunoassay for CK-BB was developed and performed exactly as described previously [ 61. In brief, antisera were raised in sheep by ~munizing with CK-BB purified from human brain. Purified CK-BB was also used for the standard curve (see Fig. 1, closed circles) and for BoftonHunter iodination with 1251.The sample volume used was 100 ~1. The mean value for healthy volunteers was about 3.5 pg/l, with the 95th percentile being about 6.0 pg/l. Crossreactivity was assessed by assaying purified myocardial CK-MB and comparing the concentrations of CK-MB and CK-BB required to displace 50% of the initi~ly-bound tracer (BIB, = 0.50).
a.1
1
10
102
103
1W
CK (ng/tube) Fig. 1. Standard curve for CK-BB and crossreactivity fore 1 ngftube corresponds to 10 ng/ml or 10 fig/l.
of cardiac
CK-MB.
Sample
volume
= 100 pl; there-
31
Results Fig. 1 shows a typical standard curve (closed circles) and the curve obtained with myocardial CK-MB (open circles). Note that the curves are more or less parallel and that the concentration of CK-MB required to achieve B/B, = 0.50 was almost lOO-fold greater than for CK-BB. The calculated crossreactivity, then, of CK-MB is (1.3/110) X 100 = 1.2%, a surprisingly low figure indicating a uniquely high degree of specificity for CK-BB. The cardiac surgery patient data are illustrated in Fig. 2, where the incremental elevations in CK-BB concentration over baseline for each post-operative day are plotted. The mean baseline value was 3.7 pg/l with a standard deviation of 1.0 and a range of 1.8-5.8. The peak increase in concentration occurred on the day of surgery, the average elevation being 22.4 (range = 7.943.7) or about a 6-fold increase over the mean baseline. The serum CK-BB concentration then fell off rapidly, so that there was little or no elevation the 3rd or 4th post-operative day.
34 32 30 28 ki i 26ti em 246 B = s 1
22zo18-
? 5 z
16-
$
12-
2
lo-
2
! (17)
\
\
\
\
\ \ \
14-
\ (47) a \ \ 1.
‘\\ \
8~
d
61 4c
I 0
141) \ ‘\
d (35) 1
2
‘-4 3
135) 4
DAYS AFTER SURGERY Fig. 2. Changes in serum CK-BB concentration up/l) following cardiac surgery. Mean incremental changes are plotted on the ordinate as open circles; vertical bars represent one standard deviation and figures in parentheses represent number of observations for each day.
32
Discussion
The patient results clearly indicate a sharp, brief rise in CK-33 concentration which occurs early in the post-operative period. This could be simply a reflection of a large increase in total serum creatine kinase activity following cardiac and other surgery, with increases in all three isoenzymes. It is known that total CK and CK-MM change in parallel in patients with a variety of disorders [7]. We measured total serum CK enzymatic activity and CK-MM by RIA in most of the serum specimens from the cardiac surgery patients. Though incomplete, this data shows clearly that both total CK activity and CR-MM concentration rose later than CK-BB, usually peaking on the second postoperative day, and remained elevated longer, often still in the abnormal range on the 5th day. This finding suggests that the changes in CK-BB are specific and are not related merely to overall surgical trauma or whatever other factors may contribute to the marked changes in total CK activity typically observed after surgery. In addition to the 47 reported here, we investigated several other patients who exhibited a different pattern. Four patients from the same cardiac surgery service, attended by the same group of physicians, but who underwent thoracotomy without cardiopulmonary bypass for other purposes such as lobectomy or pacemaker placement, had little or no change in CK-BB concentration even though they had typical postoperative changes in total CK activity and CK-MM concentration. The changes in CK-BB condensation we observed seem to be related to cardiorespiratory bypass and/or to surgical trauma to the heart. We hope to learn more about this through a prospective study in progress. Several other questions are raised by the data presented here. Firstly, does the myocardium contain CK-BB? There have been several tissue studies in which CK-BB was found in cardiac tissue although most workers have not detected CK-BB in heart. These conflicting results may be due to methodological insensitivity where CK-BB was not found, or due to artefacts where CR-BB was detected. Another possibility is that some recombination of subunits occurs during handling of the tissue preparations. Secondly, is CK-BB released from the myocardium following cellular injury? Several recent reports support the possibility that CK-BB can be, in fact, released from cardiac tissue. Vladutiu et al, [S] found (a) CK-BB, averaging 10% of total CK activity, in both coronary sinus and mixed peripheral blood taken during aortocoronary bypass surgery, (b) that CK-BB was higher in coronary sinus blood than in the corresponding peripheral blood, (c) that CK-BB was higher in patients who had had an acute myocardid infarction than in those who did not, and, (d) that fresh biopsies of ventricle contained CK-BB up to 8% of the total CK. Morgan et al. [9] studied 15 patients after coronary artery bypass surgery. All 15 had trace quantities of CK-BB in the serum and larger amounts in chest tube drainage fluid. Coolen et al. [lo] reported elevated serum CK-BB concentration in patients who had had open heart surgery. Mercer [ 11 J found increased serum CK-BB in 22% of critical-care patients for whom CK-MB determinations had been requested, as well as in patients with prolonged atrial fibrillation, congestive heart failure and cardiomyopathy.
33
Itano [ 121, in reporting 16 cases of patients with elevated CK-BB, considered cerebral anoxia or infarct to be the cause of most of these. However, 8 of these patients had cardiac arrest or ventricular fibrillation and one had congestive heart failure. In a letter [13], Elkins mentioned CK-BB elevations in specimens from coronary care unit patients. Zsigmond et al. [14] reported that 14% of CK in cardiac tissue obtained at autopsy was CK-BB. Jockers-Wretou and Pfeiderer [ 21 found only O-2% CK-BB in cardiac tissue using an immunological method. These studies suggest that myocardium may indeed contain CK-BB which can be released into the circulation in response to cardiac damage or insult. The elevations in CK-BB we report here may reflect insult to the myocardium directly through surgical manipulation and/or indirectly due to cardiorespiratory bypass. Preliminary data we have collected showing up to 15-fold increases in CK-BB in patients with myocardial infarction further support the hypothesis that CK-BB may be released from heart. It is well known that CK-MB rises following cardiac surgery with or without perioperative infarction. While it is possible that our assay is not as specific as it appears from the crossreactivity studies we have performed [6] and that we may be actually detecting changes in CK-MB or a combination of CK-MB and CK-BB, we feel that we are indeed measuring CK-BB. Much of the data from the literature that we have discussed above was generated using electrophoresis. Confusion between CK-MB and CK-BB is unlikely when this technique is used correctly. Therefore, the hypothesis that CK-BB release is associated with myocardial damage seems to have growing support. References 1 Wurzburg, II.. Hennrich, N.. Orth. H.-D., Lang, H.. PreIIwitz, W., Neumeier. D.. Knedel. M. and Rick, W. (1977) J. CIin. Chem. Clin. Biochem. 15.131-137 2 Jockers-Wretou, E. and Pfleiderer. G. (1975) Clin. Chim. Acta 58. 223-232 3 Roberts, R., Parker, C.S. and Sobel. B.E. (1977) Lancet ii, 319-322 4 WiRerson. J.T.. Stone, M.J., Ting. R.. Mukherjee, A., Games-Sanchez. C.E.. Lewis. P. and Hersh. L.B. (1977) Proc. NatI. Acad. Sci. U.S.A. 74.1711-1715 5 Neumeier, D. and Hofstetter. R. (1977) Chn. Chim. Acta 79.107-113 6 Zweig, M.H., Van Steirteghem, A.C. and Schechter, A.N. (1978) Chn. Chem. 24.422-428 7 Van Steirteghem. AC.. Zweig. M.H. and Schechter, A.N. (1978) Clin. Chem. 24.414421 8 VIadutiu, A.O.. Schachner. A., Schaefer, P.A.. Schimert. G., Laios. T.Z., Lee, A.B. and Siegel, J.H. (1977) CIin. Chim. Acta 75,467473 9 Morgan, J.. FenneIL W., Bozyk. M.E. and Cohen, L. (1977) Clm. Res. 25. 239A (Abstract) 10 Coolen. R.B., Pragay. D.A. and ChiIcote; M.E. (1975) CU. Chem. 21.976 (Abstract) 11 Mercer. D.W. (1977) Chn. Chem. 23.611-612 (Letter) 12 Itano. M. (1976) Am. J. CIin. Pathol. 65.351-355 13 Elkins, B.N. (1977) CIin. Chem. 23.1510 (Letter) 14 Zsigmond. E.K.. Starkweather, W.H.. Duboff, G.S. and Flynn. K.A. (1972) Anesth. Anal. 51. 827840
Clinica Chimica Acta, 93 (1979) 29-33 0 Elsevier/North-Holland Biomedical Press
CCA 10103
INCREASED CK-BB CONCENTRATIONS CARDIAC SURGERY
MARK H. ZWEIG * and ANDRfi Clinical Pathology Department, MD 20014 (U.S.A.) (Received
October
20th,
IN SERUM FOLLOWING
C. VAN STEIRTEGHEM
**
Clinical Center, National Institutes of Health, Bethesda,
1978)
Summary We measured the BB isoenzyme of creatine kinase by a specific radioimmunoassay in the serum of 47 patients following cardiac surgery. A sharp increase in CK-BB occurred immediately after surgery, with rapid return to baseline by the fourth post-operative day. This data, along with other reports in the literature, suggest that CK-BB is released into the circulation following myocardial insult.
Introduction Serum creatine kinase (CK) activity has received much attention recently by those interested in the diagnosis and management of myocardial infarction. Most recently, serial measurements have been used in attempts to estimate infarct size and to assess efficacy in interventions designed to limit infarct extension and to reduce complications. Many claims have come forth regarding the specificity and sensitivity of the MB or “myocardial” isoenzyme of CK for the diagnosis of acute myocardial infarction. As a result, there is widespread use of electrophoretic and anion exchange column chromatographic methods for separating and measuring CK-MB enzymatic activity in serum. Because of various problems with these methods, immunochemical approaches such as immunoinhibition, immunoprecipitation and radioimmunoassay have been applied to the measurement of CK-MB [l-5]. None of the radioimmunoassays is specific for the MB isoenzyme. All claim to measure B subunit activity in both the MB and BB isoenzymes. The assumption is made that the BB isoenzyme is negligible or absent, in spite of numerous reports to the contrary [ 61. Furthermore, CK-MB is still widely considered specific for acute myocar* To whom correspondence should be addressed. ** Present address: Clinical Chemistry Service, Vrije Universiteit Brussel. Universitair Ziekenhuis SintPieter. Hoogstreat, B-1000 Brussels, Belgium.
30
dial infarction even though it is found to be increased in serum in a wide variety of clinical situations. We have developed a sensitive radioimmunoassay for the BB isoenzyme which appears specific, havong only 1% crossrea~tivity with CK-MB [6]. In the course of measuring CK-BB in the serum of unselected patients, we found 20-fold elevations of CK-BB in two patients who had undergone cardiac surgery. Because of this finding we looked more closely at patients having cardiac surgery and found that virtually all had elevated serum CK-BB concentrations immediately after surgery, persisting for several days. Methods Forty-seven patients undergoing cardiac surgery, usually with cardiopulmonary bypass, were followed. The surgical procedures included coronary artery bypass graft, valve replacement, septal defect repair, ventricular myectomy, etc. Where possible, serum obtained post-operatively on the day of surgery and daily thereafter was assayed. Baseline values were established for each patient by assaying preoperative serum or post-operative serum five or more days after surgery. The radioimmunoassay for CK-BB was developed and performed exactly as described previously [ 61. In brief, antisera were raised in sheep by ~munizing with CK-BB purified from human brain. Purified CK-BB was also used for the standard curve (see Fig. 1, closed circles) and for BoftonHunter iodination with 1251.The sample volume used was 100 ~1. The mean value for healthy volunteers was about 3.5 pg/l, with the 95th percentile being about 6.0 pg/l. Crossreactivity was assessed by assaying purified myocardial CK-MB and comparing the concentrations of CK-MB and CK-BB required to displace 50% of the initi~ly-bound tracer (BIB, = 0.50).
a.1
1
10
102
103
1W
CK (ng/tube) Fig. 1. Standard curve for CK-BB and crossreactivity fore 1 ngftube corresponds to 10 ng/ml or 10 fig/l.
of cardiac
CK-MB.
Sample
volume
= 100 pl; there-
31
Results Fig. 1 shows a typical standard curve (closed circles) and the curve obtained with myocardial CK-MB (open circles). Note that the curves are more or less parallel and that the concentration of CK-MB required to achieve B/B, = 0.50 was almost lOO-fold greater than for CK-BB. The calculated crossreactivity, then, of CK-MB is (1.3/110) X 100 = 1.2%, a surprisingly low figure indicating a uniquely high degree of specificity for CK-BB. The cardiac surgery patient data are illustrated in Fig. 2, where the incremental elevations in CK-BB concentration over baseline for each post-operative day are plotted. The mean baseline value was 3.7 pg/l with a standard deviation of 1.0 and a range of 1.8-5.8. The peak increase in concentration occurred on the day of surgery, the average elevation being 22.4 (range = 7.943.7) or about a 6-fold increase over the mean baseline. The serum CK-BB concentration then fell off rapidly, so that there was little or no elevation the 3rd or 4th post-operative day.
34 32 30 28 ki i 26ti em 246 B = s 1
22zo18-
? 5 z
16-
$
12-
2
lo-
2
! (17)
\
\
\
\
\ \ \
14-
\ (47) a \ \ 1.
‘\\ \
8~
d
61 4c
I 0
141) \ ‘\
d (35) 1
2
‘-4 3
135) 4
DAYS AFTER SURGERY Fig. 2. Changes in serum CK-BB concentration up/l) following cardiac surgery. Mean incremental changes are plotted on the ordinate as open circles; vertical bars represent one standard deviation and figures in parentheses represent number of observations for each day.
32
Discussion
The patient results clearly indicate a sharp, brief rise in CK-33 concentration which occurs early in the post-operative period. This could be simply a reflection of a large increase in total serum creatine kinase activity following cardiac and other surgery, with increases in all three isoenzymes. It is known that total CK and CK-MM change in parallel in patients with a variety of disorders [7]. We measured total serum CK enzymatic activity and CK-MM by RIA in most of the serum specimens from the cardiac surgery patients. Though incomplete, this data shows clearly that both total CK activity and CR-MM concentration rose later than CK-BB, usually peaking on the second postoperative day, and remained elevated longer, often still in the abnormal range on the 5th day. This finding suggests that the changes in CK-BB are specific and are not related merely to overall surgical trauma or whatever other factors may contribute to the marked changes in total CK activity typically observed after surgery. In addition to the 47 reported here, we investigated several other patients who exhibited a different pattern. Four patients from the same cardiac surgery service, attended by the same group of physicians, but who underwent thoracotomy without cardiopulmonary bypass for other purposes such as lobectomy or pacemaker placement, had little or no change in CK-BB concentration even though they had typical postoperative changes in total CK activity and CK-MM concentration. The changes in CK-BB condensation we observed seem to be related to cardiorespiratory bypass and/or to surgical trauma to the heart. We hope to learn more about this through a prospective study in progress. Several other questions are raised by the data presented here. Firstly, does the myocardium contain CK-BB? There have been several tissue studies in which CK-BB was found in cardiac tissue although most workers have not detected CK-BB in heart. These conflicting results may be due to methodological insensitivity where CK-BB was not found, or due to artefacts where CR-BB was detected. Another possibility is that some recombination of subunits occurs during handling of the tissue preparations. Secondly, is CK-BB released from the myocardium following cellular injury? Several recent reports support the possibility that CK-BB can be, in fact, released from cardiac tissue. Vladutiu et al, [S] found (a) CK-BB, averaging 10% of total CK activity, in both coronary sinus and mixed peripheral blood taken during aortocoronary bypass surgery, (b) that CK-BB was higher in coronary sinus blood than in the corresponding peripheral blood, (c) that CK-BB was higher in patients who had had an acute myocardid infarction than in those who did not, and, (d) that fresh biopsies of ventricle contained CK-BB up to 8% of the total CK. Morgan et al. [9] studied 15 patients after coronary artery bypass surgery. All 15 had trace quantities of CK-BB in the serum and larger amounts in chest tube drainage fluid. Coolen et al. [lo] reported elevated serum CK-BB concentration in patients who had had open heart surgery. Mercer [ 11 J found increased serum CK-BB in 22% of critical-care patients for whom CK-MB determinations had been requested, as well as in patients with prolonged atrial fibrillation, congestive heart failure and cardiomyopathy.
33
Itano [ 121, in reporting 16 cases of patients with elevated CK-BB, considered cerebral anoxia or infarct to be the cause of most of these. However, 8 of these patients had cardiac arrest or ventricular fibrillation and one had congestive heart failure. In a letter [13], Elkins mentioned CK-BB elevations in specimens from coronary care unit patients. Zsigmond et al. [14] reported that 14% of CK in cardiac tissue obtained at autopsy was CK-BB. Jockers-Wretou and Pfeiderer [ 21 found only O-2% CK-BB in cardiac tissue using an immunological method. These studies suggest that myocardium may indeed contain CK-BB which can be released into the circulation in response to cardiac damage or insult. The elevations in CK-BB we report here may reflect insult to the myocardium directly through surgical manipulation and/or indirectly due to cardiorespiratory bypass. Preliminary data we have collected showing up to 15-fold increases in CK-BB in patients with myocardial infarction further support the hypothesis that CK-BB may be released from heart. It is well known that CK-MB rises following cardiac surgery with or without perioperative infarction. While it is possible that our assay is not as specific as it appears from the crossreactivity studies we have performed [6] and that we may be actually detecting changes in CK-MB or a combination of CK-MB and CK-BB, we feel that we are indeed measuring CK-BB. Much of the data from the literature that we have discussed above was generated using electrophoresis. Confusion between CK-MB and CK-BB is unlikely when this technique is used correctly. Therefore, the hypothesis that CK-BB release is associated with myocardial damage seems to have growing support. References 1 Wurzburg, II.. Hennrich, N.. Orth. H.-D., Lang, H.. PreIIwitz, W., Neumeier. D.. Knedel. M. and Rick, W. (1977) J. CIin. Chem. Clin. Biochem. 15.131-137 2 Jockers-Wretou, E. and Pfleiderer. G. (1975) Clin. Chim. Acta 58. 223-232 3 Roberts, R., Parker, C.S. and Sobel. B.E. (1977) Lancet ii, 319-322 4 WiRerson. J.T.. Stone, M.J., Ting. R.. Mukherjee, A., Games-Sanchez. C.E.. Lewis. P. and Hersh. L.B. (1977) Proc. NatI. Acad. Sci. U.S.A. 74.1711-1715 5 Neumeier, D. and Hofstetter. R. (1977) Chn. Chim. Acta 79.107-113 6 Zweig, M.H., Van Steirteghem, A.C. and Schechter, A.N. (1978) Chn. Chem. 24.422-428 7 Van Steirteghem. AC.. Zweig. M.H. and Schechter, A.N. (1978) Clin. Chem. 24.414421 8 VIadutiu, A.O.. Schachner. A., Schaefer, P.A.. Schimert. G., Laios. T.Z., Lee, A.B. and Siegel, J.H. (1977) CIin. Chim. Acta 75,467473 9 Morgan, J.. FenneIL W., Bozyk. M.E. and Cohen, L. (1977) Clm. Res. 25. 239A (Abstract) 10 Coolen. R.B., Pragay. D.A. and ChiIcote; M.E. (1975) CU. Chem. 21.976 (Abstract) 11 Mercer. D.W. (1977) Chn. Chem. 23.611-612 (Letter) 12 Itano. M. (1976) Am. J. CIin. Pathol. 65.351-355 13 Elkins, B.N. (1977) CIin. Chem. 23.1510 (Letter) 14 Zsigmond. E.K.. Starkweather, W.H.. Duboff, G.S. and Flynn. K.A. (1972) Anesth. Anal. 51. 827840