European Heart Journal (1992) 13, 188-193
Lack of indication of myocardial cell damage after myocardial ischaemia in patients with severe stable angina K. N0RREGAARD-HANSEN*, K. EGSTRUP*, J. R. NIELSEN*, P. J. JoRGENSENf, P. H. PETERSENf, T. HAGHFELT* AND M. H0RDERf
* Departments of Cardiology and ^Clinical Chemistry, Odense University Hospital, Denmark KEY WORDS: Angina pectoris, myocardial ischaemia, silent ischaemia, exercise testing, myoglobin, creatine kinase, isoenzyme MB, serum. To evaluate myocardial cell damage in relation to spontaneous and exercise-induced ischaemia, release of myoglobin, creatine kinase (CK) and its isoenzyme MB (CK-MB) into the serum was estimated in 10 patients with severe stable angina. All patients had a positive exercise test, significant stenosis of one or more of the main coronary arteries and more than five ischaemic attacks per week. ST-segment monitoring was performedfor 36 h. During the last 24 h of that period (period A) serial blood samples were analysed for myoglobin, CK and CK-MB using sensitive assays. Three days later (period B) the patients performed an exercise test at 0815 h, with ST-segment monitoring and blood sampling carried out as described for period A. During period A, 47 ischaemic episodes (100% silent) with a total duration of 599 min were noted in four patients. Forty-seven ischaemic episodes (94%> silent) with a total duration of804 min, were observed in seven patients during period B. Release of myoglobin, CK, and CK-MB did not increase in relation either to spontaneous or exercise-induced ischaemia. Thus even frequent and prolonged episodes of transient myocardial ischaemia (symptomatic or asymptomatic) in patients with severe stable angina pectoris does not seem to cause irreversible myocardial damage. Introduction
Necrosis in acute myocardial infarction (AMI) is the major cause of release of myocardial enzymes and myoglobin into the circulation'1"31. Even in patients with unstable or variant angina, minor release of myocardial enzymes has been observed, indicating that limited myocardial necrosis may also take place in these conditions'4"61. In contrast, biochemical evidence of myocardial necrosis has not been observed in stable angina in relation to exercise-induced ischaemia'7'81. However, studies that have utilized ambulatory ST-segment monitoring have recently shown that symptomatic ischaemic episodes only represent a minority of total ischaemic activity in patients with stable angina and that transient silent ischaemic episodes may be very frequent, prolonged and with high grade ST-segment changes'91. The total ischaemic burden may therefore be high and even exceed the duration of ischaemia which has been observed to induce myocardial necrosis in animal studies'101. Furthermore, animal studies have shown that repetitive ischaemic episodes of shorter duration may have a cumulative effect which can lead to myocardial necrosis'10"1. We therefore decided to assess the markers of myocardial necrosis i.e. release of myoglobin, CK and CK-MB into the systemic circulation in relation to symptomatic and silent ischaemic episodes and to exercise-induced ischaemia in patients with severe stable angina pectoris. Patients and methods
Individual patients' characteristics are given in Table 1. Ten male patients with chronic stable angina were Submitted for publication on 2 July 1990, and in revised form 8 February 1991. Concspondenet: Knud Norregaard-Hansen, Klovervtcnget 26 C, DK-5000 Odense, Denmark 0195-668X 92 020188 + 06 $03,00 0
included; their mean age was 54-1 (range: 40-65) years and the mean duration of angina was 51 (range: 2-12) years. Three had had a previous AMI more than 3 months before inclusion. Selective coronary arteriography (CAG) performed within a week from the start of the study showed that two patients had one-vessel disease,fivehad two-vessel disease (including one with left main stem (LM) stenosis) and three had three-vessel disease (including two with LM stenosis). Collateral circulation was demonstrated in nine patients. The ejection fraction was normal in eight patients (>60%) and reduced in two ( 1 mm measured 80 ms after the J-point. An episode had to last for at least 1 min and any two episodes has to be separated by at least 2 min of return to the ECG to baseline. To evaluate symptomatic ischaemic episodes all patients kept a detailed diary and were instructed to activate the event button on the tape recorder when they were experiencing anginal chest pain. The number, duration and maximal ST-changes with symptomatic and asymptomatic ischaemic episodes were noted for each tape.
Exercise testing A maximal symptom-limited bicycle exercise stress test was performed about one month before the study and during period B. The test was terminated in cases of severe anginal pain, complex ventricular arrhythmias or hypotension. A 12-lead precordial mapping system was used to improve the definition of the area with maximal STsegment depression"8'. The test started with a workload of 30 W which was increased every min by 10 W. The test was considered positive if the patient developed typical anginal pain and significant ST-segment depression with the same criteria as during ST-segment monitoring.
190 K. Nerregaard-Hansen et al.
Table 2
Distribution of spontaneous episodes of ischaemia during periods A and B in patients with silent or symptomatic (*) ischaemia
Patient number
A
B
No. of ischaemic episodes and duration (number of min) in the time intervals 20-00
1 2 3 10
1(24) — — 4(100)
1 2 3 5 8 9 10
3(78) — — — — — 7(74)
00-04
04-08
08-12
12-16
16-20
20-00
00-04
04-08
— — 2(31) — — — — — — —
1(29) 1(12) — 4(21) — — 1(23) 1(38) — — 5(102)
3(40) 1(18) 1(37) 5(70) — — — 1(41) — — 6(73)
1(19) — — 4(70)
3(38) 1(12) — 4(33) — — — 1(31)* 1 (27)* I (55)* 4(40)
1 (22) 1(12) — 3(21) — 1(12) 1(15) 1 (28) — 2(14) 1 (2)
— — — 9(58) — 1(15) — — — —
— — — 1(2) — — — — — 1 (9)
Mathematical and statistical methods Non-parametric two-way analysis of variance (Friedman's test for related samples) was used to compare the levels of myoglobin, CK and CK-MB for the eight sampling times for each period (A and B). If the null hypothesis was rejected by this test, multiple comparisons were carried out by use of Friedman's rank sums'"1. To analyse the influence of exercise-induced ischaemia on relase of myoglobin, CK. and CK-MB the levels of these markers during period B (the day of exercise testing) were compared with period A (the day without exercise testing) by non-parametric two-way analysis of variance119'20'. In order to analyse the influence of spontaneous ischaemia (symptomatic or silent) on release of myoglobin, CK or CK-MB, the serum concentrations of these markers among individuals with spontaneous ischaemia were compared with those of patients without ischaemia and with the reference limits found in apparent healthy noncardiac subjects. Furthermore, in order to reduce the large differences between the levels of S-myoglobin, S-CK and S-CK-MB of the patients, the data were normalized. The normalization within the individual patient was performed by dividing each value by the mean of all eight measurements obtained during each period. As a reference interval for these fractional concentrations the 95% confidence interval (mean ± 2 pooled SD) of the six patients without episodes of ischaemia during period A (Table 2) was calculated.
Results ST-SEGMENT MONITORING AND EXERCISE TESTING
All patients had a positive exercise test with significant ST-depression (mean: 2-8 mm, range: 1-7 mm) and the mean maximum workload was 1120W (range: 80-170) (Table 1). Table 2 shows the distribution of spontaneous ischaemic episodes during periods A and B. During period A, a total of 47 episodes (all silent) were noted in four patients. The total duration for all episodes of all patients was 599 min (range 37-336). Monitoring in period B detected 47 episodes of ischaemia obtained in
1(28) — — 1(11) — — 6(74)
Total 10(172) 4(54) 1(37) 32(336) 4(106) 2(21) 2(38) 5(149) 1(27) 3(69) 30(394)
seven patients and 91% were asymptomatic. The total duration of ischaemia for all episodes of all patients was 804 min (range 21-394). MYOGLOBIN
The median concentration and inter quartile range of Smyoglobin for each sampling time of periods A and B are shown in Fig. 1 (upper panel). During both periods, Smyoglobin had a significant circadian variation, with highest values in the morning and lowest values in the late afternoon (P005). The fractional concentration of Smyoglobin in the patients with spontaneous ischaemia during period A (No 1,2,3,10) and period B (1,2, 3, 5, 8, 9, 10) was largely within the reference interval (Fig. 3: upper panel). CK
In Fig. 1 (middle panel), the median activity and inter quartile range of S-CK during periods A and B is displayed. During both periods a minor but significant variation in S-CK was found during the day and with highest activity at 1900 h (P = 0021). No difference in S-CK level was found between period B (after exercise testing) and period A (P = 0-084). The median and inter quartile ranges in S-CK for patients with and without ischaemia during period A are shown in Fig. 2 (middle panel). The values of the patients with ischaemic attacks did not exceed the upper reference limit of non-cardiac patients, and no difference between patients with and without ischaemia were found (P>005). During period B, two patients (Nos 1 and 2) had minor peak values exceeding the upper 95% reference interval of the fractional
No cell damage after myocardial ischaemia? 191
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Figure 1 The median and inter quartile range of S-myoglobin (upper panel) S-CK (middle panel) and S-CK-MB (lower panel) in 10 patients with stable angina pectoris at eight sampling times during 24 h at minimal activity (• = period A) and during 24 h following exercise at 0815 h ( • = period B).
activities (Fig. 3: middle panel) at 1200 h, but these peaks had no relation to the occurrence of ischaemic episodes.
I
Figure 2 The median and inter quartile range of S-myoglobin (upper panel) S-CK (middle panel) and S-CK-MB (lower panel) in four patients with and six patients without spontaneous ischaemia during period A. The upper reference limit (dotted line) for apparently healthy non-cardiac subjects is indicated for S-myoglobin and S-CK-MB. For S-CK this limit is 270 U 11 '. * = ischaemia; D = n o ischaemia.
S-CK-MB exceeding the upper reference limit of healthy subjects, but his S-CK-MB curve showed no changes Fig. 1 (lower panel) displays the median concentration throughout the day compatible with myocardial release of and inter quartile range of S-CK-MB during periods A the enzyme (Fig. 2: lower panel); this patient had a correand B. No circadian variation was found during the two spondingly high level of CK-MB during period B. At each periods (P> 0-45), and the level of period B (after exercise separate sampling time no difference between the S-CKtesting) was not higher than the level of j>eriod A (P = MB values of patients with and without ischaemia could 0-69). During period A, one patient (no. 3) had values of be found (P>005) (Fig. 2: lower panel). During both CK-MB
192 K. Nerregaard-Hansen etal.
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Figure 3 Fractional concentration of myoglobin (upper panel), CK (middle panel) and CK-M B (lower panel) in four patients with spontaneous ischaemic attacks during period A(V — V) (Nos 1,2,3,10) and in seven patients with spontaneous attacks during period B (C C) (Nos 1, 2, 3, 5, 8, 9, 10). The shaded area represents the 95% reference interval (mean ±2 pooled SD) calculated from the six patients without spontaneous ischaemia during period A.
periods the fractional concentration of S-CK-MB of patients with spontaneous ischaemia was largely within the reference interval (Fig. 3: lower panel). Discussion
This study shows that in patients with severe stable angina no detectable amount of myoglobin, CK and CK-
MB is released into the peripheral circulation in relation to prolonged and repetitive spontaneous ischaemic episodes or exercise-induced ischaemia. In several animal studies periods of coronary occlusion above 20-30 min have resulted in myocardial cell necrosis with release of myocardial enzymes, and briefer if repetitive (5-15 min) episodes may also lead to myocardial necrosis'10'. The total ischaemic burden observed in some of the patients in our study exceeds the ischaemic burden provoked in these animal studies'10'. In these experiments, however, ischaemia was induced by total occlusion of one of the coronary arteries, whereas ischaemia in stable angina is normally related to a partial reduction of coronary blood flow and to increased myocardial oxygen demands, but not to coronary occlusion. Thus, Block et al. (1983) found that a 50% reduction in left main coronary artery diameter did not lead to myocardial necrosis or release of myoglobin'21'. Furthermore the collateral circulation which is often present in patients with chronic stable angina may partly protect ischaemic myocardium'22'. Assessment by measurement in peripheral blood samples of increased release of intracellular proteins from myocardial cells instead of samples from the coronary sinus may be less sensitive, because of a dilution effect'23'. Sylven et al. (1984) measured myoglobin in the coronary sinus blood after paced-induced angina and they suggested a minor release of myoglobin into the coronary circulation124'. Blood sampling from the coronary sinus however would not be possible over long study periods as in this study. To compensate for the dilution effect we have used highly sensitive analytical methods. For the analyses of CK-MB a recently developed immunoassay with low imprecision and low detection limit was used1'71. The immunoinhibition method most often used for CK isoenzyme analysis in patients suspected of AMI is not suitable because of high imprecision in the low range1'1. Clinical studies have documented that release of intracardial proteins, such as myoglobin, CK and CK-M B into the circulation is a sensitive indicator of myocardial necrosis in patients with AMI'1"3'. Minor, but significant release has also been shown to take place in some patients with unstable angina pectoris'451. It is most likely that this release reflects myocardial cell death and not leakage from reversibly damaged cells. Subendocardial necrosis in unstable angina patients has indeed been proved by other methods such as technetium-99m pyrophosphate myocardial scintigraphy125'. The fact that no increased release of myoglobin, CK and CK-MB could be detected in our patients therefore seems to exclude myocardial necrosis. Ourfindingsare in accordance with those of Baadsgaard and Schmidt (1984) and Marmor et al. (1978) who found no release of myoglobin and creatine kinase isoenzymes in patients with stable angina pectoris after exercise testing"-81. Neither did Bagger et al. (1982) find release followed by pacing induced ischaemia'23'. In conclusion prolonged and repetitive spontaneous symptomatic or asymptomatic ischaemia and exerciseinduced ischaemia in stable angina pectoris are not associated with irreversible myocardial cell damage as measured from release of myoglobin, CK or CK-MB.
No cell damage after myocardial ischaemia? 193
This study was kindly supported by a grant from 'Guldsmed AL and D. Rasmussensmindelegat'.
References [1] Gerhardt W, Waldenstrem J, Herder M et al. Crcatine kinasc and crcatine kinase B-subunit activity in serum in cases of suspected myocardial infarction. Clin Chem 1982; 28: 277-83. [2] Stone MJ, Waterman MR, Poliner LR, Templeton GH, Buja LM, Willerson JT. Myoglobinemia is an early and quantitative index of acute myocardial infarction. Ange'iologie 1978; 29: 386-92. [3] Nerregaard-Hansen K, Hyltoft Petersen P, Hangaard J, Simonscn EE, Rasmussen O, Herder M. Early observations of S-myoglobin in the diagnosis of acute myocardial infarction. The influence of discrimination limit, analytical quality, patient's sex and prevalence of disease. Scand J clin Lab Invest 1986; 46: 561-9. [4] Thygesen K, Herder M, Krell L, Hyltoft Petersen P, Haghfelt T. Creatine kinase and creatine kinase B-subunit in stable and unstable angina pectoris. Eur J clin Invest 1984; 16: 1-4. [5] Hoberg E, Katus HA, Diederich KW, Kubler W. Myoglobin, creatine kinase B subunit, and myosin light chain release in patients with unstable angina. Eur Heart J 1987; 8: 989-94. [6] Biagini A, Mazzei MG, Carpeggiam C, Buzzigali G, Zuchelli G, Parodi O, L'Abbate A, Maseri A. Myocardial cell damage during attacks of vasospastic angina in the absence of persistent electrocardiographic changes. Clin Cardial 1981; 4: 315-9 [7] Baadsgaard O, Schmidt JF. Myoglobin concentration, creatine kinase, and creatine kinase subunit B activity in serum after myocardial ischemia. Scand J clin Lab Invest 1984; 44: 679-82 [8] Marmor A, Alpan G, Keidar S, Grenardier E, Palant A. The MB isoenzyme of creatine kinase as a indicator of severity of myocardial ischemia. Lancet 1978; li: 812-4. [9] Egstrup K. Transient myocardial ischemia after abrupt withdrawal of antianginal therapy in chronic stable angina. Am J Cardiol 1988,61: 1219-22. [ 10] Geft IL, Fishbein MC, Ninomiya K, Hashida J, Chaux E, Yano J el al. Intermittent brief periods of ischemia have a cumalative effect and may cause myocardial necrosis. Circulation 1982; 66/6: 1150-3 (11] Braunwald E, Kloner RA The stunned myocardium, prolonged postischemic ventricular dysfunction. Circulation 1982; 66/-6. 1146-9.
[12] Ellis AK, Little T, Masud ARZ, Liberman HA, Morris DC, Klocke FJ. Early non invasive detection of successful reperfusion in patients with acute myocardial infarction. Circulation 1988; 78: 1352-7. [13] Nerregaard-Hansen K, Nergaard-Pedersen. Rapid and sensitive radioimmunoassays for human myoglobin. Scand J clin Lab Invest 1979; 39: 525-31. [14] Gerhardt W, Waldenstrem J. Creatine kinase B-subunit activity in serum after immunoinhibition of M-subunit Activity. Clin Chem 1979; 25/7: 1274-80. [15] Gerhardt W, Waldenstrem J, Herder M, Hofvendahl S, Ljungdahl R, Beming H, Bagger P. Creatine kinase and creatine kinase B-subunit activity in cases of suspected myocardial infarction. Clin Chem 1982; 28/2: 277-83. [16] Herder M, Thygesen K, Gerhardt W, Grande P, Christiansen I, Stender S. Enzymdiagnostik ved akut myokardieinfarkt. UgeskrLacger 1989; 151/23: 1447-53. [17] Jergensen PJ, Selmer J, Herder M. Analytical evaluation of a sensitive enzyme immunoassay for creatine kinase isoenzyme MB. Clin Chem 1990; 36/8: 1502-5. [18] Egstrup K. Transient myocardial ischemia during ambulatory monitoring out of hospital in patients with chronic stable angina pectoris. Acta Med Scand 1988; 224: 311-8. [19] Hollander M, Wolfe A. Non parametric statistical methods, pp. 151-158. New York: John Wiley, 1973 [20] Wulff HR, Schlichting P Medstat: Statistical program for analysis of results of controlled therapeutic trials and other types of clinical research. Version 2.1, 1988. The Astra Group A/S, Denmark [21] Block MI, Said JW, Siegel RJ, Fishbein MC. Myocardial myoglobin following coronary artery occlusion. Am J Pathol 1983; 111:374-9. [22] Norell MS, Lyons JP, Gardener J, Layton CA, Balcon R. Protective effect of collateral vessels during coronary angioplasty. Br Heart J 1989; 62: 241-5 [23] Bagger JP, Ingerslev J, Heinsvig EM. Creatine kinase and creatine kinase subunit-B in coronary sinus blood in pacinginduced angina pectons. Scand J clin Lab Invest 1982; 42: 617-20. [24] Sylven C, Berglund B, Kaijser L. Myoglobin release in patients with angina pectons following short term ischaemia by pacing to angina. Clinical Physiology 1984; 4- 221-6 [25] Donsky MS, Curry GC, Parkey RW, Meyer SL, Bonte FJ, Platt MR, Willerson JT. Unstable Angina pectoris. Clinical, angiographic, and myocardial scintigraphic observations. Br Heart J 1976; 38- 257-63.
Lack of indication of myocardial cell damage after myocardial ischaemia in patients with severe stable angina K. N0RREGAARD-HANSEN*, K. EGSTRUP*, J. R. NIELSEN*, P. J. JoRGENSENf, P. H. PETERSENf, T. HAGHFELT* AND M. H0RDERf
* Departments of Cardiology and ^Clinical Chemistry, Odense University Hospital, Denmark KEY WORDS: Angina pectoris, myocardial ischaemia, silent ischaemia, exercise testing, myoglobin, creatine kinase, isoenzyme MB, serum. To evaluate myocardial cell damage in relation to spontaneous and exercise-induced ischaemia, release of myoglobin, creatine kinase (CK) and its isoenzyme MB (CK-MB) into the serum was estimated in 10 patients with severe stable angina. All patients had a positive exercise test, significant stenosis of one or more of the main coronary arteries and more than five ischaemic attacks per week. ST-segment monitoring was performedfor 36 h. During the last 24 h of that period (period A) serial blood samples were analysed for myoglobin, CK and CK-MB using sensitive assays. Three days later (period B) the patients performed an exercise test at 0815 h, with ST-segment monitoring and blood sampling carried out as described for period A. During period A, 47 ischaemic episodes (100% silent) with a total duration of 599 min were noted in four patients. Forty-seven ischaemic episodes (94%> silent) with a total duration of804 min, were observed in seven patients during period B. Release of myoglobin, CK, and CK-MB did not increase in relation either to spontaneous or exercise-induced ischaemia. Thus even frequent and prolonged episodes of transient myocardial ischaemia (symptomatic or asymptomatic) in patients with severe stable angina pectoris does not seem to cause irreversible myocardial damage. Introduction
Necrosis in acute myocardial infarction (AMI) is the major cause of release of myocardial enzymes and myoglobin into the circulation'1"31. Even in patients with unstable or variant angina, minor release of myocardial enzymes has been observed, indicating that limited myocardial necrosis may also take place in these conditions'4"61. In contrast, biochemical evidence of myocardial necrosis has not been observed in stable angina in relation to exercise-induced ischaemia'7'81. However, studies that have utilized ambulatory ST-segment monitoring have recently shown that symptomatic ischaemic episodes only represent a minority of total ischaemic activity in patients with stable angina and that transient silent ischaemic episodes may be very frequent, prolonged and with high grade ST-segment changes'91. The total ischaemic burden may therefore be high and even exceed the duration of ischaemia which has been observed to induce myocardial necrosis in animal studies'101. Furthermore, animal studies have shown that repetitive ischaemic episodes of shorter duration may have a cumulative effect which can lead to myocardial necrosis'10"1. We therefore decided to assess the markers of myocardial necrosis i.e. release of myoglobin, CK and CK-MB into the systemic circulation in relation to symptomatic and silent ischaemic episodes and to exercise-induced ischaemia in patients with severe stable angina pectoris. Patients and methods
Individual patients' characteristics are given in Table 1. Ten male patients with chronic stable angina were Submitted for publication on 2 July 1990, and in revised form 8 February 1991. Concspondenet: Knud Norregaard-Hansen, Klovervtcnget 26 C, DK-5000 Odense, Denmark 0195-668X 92 020188 + 06 $03,00 0
included; their mean age was 54-1 (range: 40-65) years and the mean duration of angina was 51 (range: 2-12) years. Three had had a previous AMI more than 3 months before inclusion. Selective coronary arteriography (CAG) performed within a week from the start of the study showed that two patients had one-vessel disease,fivehad two-vessel disease (including one with left main stem (LM) stenosis) and three had three-vessel disease (including two with LM stenosis). Collateral circulation was demonstrated in nine patients. The ejection fraction was normal in eight patients (>60%) and reduced in two ( 1 mm measured 80 ms after the J-point. An episode had to last for at least 1 min and any two episodes has to be separated by at least 2 min of return to the ECG to baseline. To evaluate symptomatic ischaemic episodes all patients kept a detailed diary and were instructed to activate the event button on the tape recorder when they were experiencing anginal chest pain. The number, duration and maximal ST-changes with symptomatic and asymptomatic ischaemic episodes were noted for each tape.
Exercise testing A maximal symptom-limited bicycle exercise stress test was performed about one month before the study and during period B. The test was terminated in cases of severe anginal pain, complex ventricular arrhythmias or hypotension. A 12-lead precordial mapping system was used to improve the definition of the area with maximal STsegment depression"8'. The test started with a workload of 30 W which was increased every min by 10 W. The test was considered positive if the patient developed typical anginal pain and significant ST-segment depression with the same criteria as during ST-segment monitoring.
190 K. Nerregaard-Hansen et al.
Table 2
Distribution of spontaneous episodes of ischaemia during periods A and B in patients with silent or symptomatic (*) ischaemia
Patient number
A
B
No. of ischaemic episodes and duration (number of min) in the time intervals 20-00
1 2 3 10
1(24) — — 4(100)
1 2 3 5 8 9 10
3(78) — — — — — 7(74)
00-04
04-08
08-12
12-16
16-20
20-00
00-04
04-08
— — 2(31) — — — — — — —
1(29) 1(12) — 4(21) — — 1(23) 1(38) — — 5(102)
3(40) 1(18) 1(37) 5(70) — — — 1(41) — — 6(73)
1(19) — — 4(70)
3(38) 1(12) — 4(33) — — — 1(31)* 1 (27)* I (55)* 4(40)
1 (22) 1(12) — 3(21) — 1(12) 1(15) 1 (28) — 2(14) 1 (2)
— — — 9(58) — 1(15) — — — —
— — — 1(2) — — — — — 1 (9)
Mathematical and statistical methods Non-parametric two-way analysis of variance (Friedman's test for related samples) was used to compare the levels of myoglobin, CK and CK-MB for the eight sampling times for each period (A and B). If the null hypothesis was rejected by this test, multiple comparisons were carried out by use of Friedman's rank sums'"1. To analyse the influence of exercise-induced ischaemia on relase of myoglobin, CK. and CK-MB the levels of these markers during period B (the day of exercise testing) were compared with period A (the day without exercise testing) by non-parametric two-way analysis of variance119'20'. In order to analyse the influence of spontaneous ischaemia (symptomatic or silent) on release of myoglobin, CK or CK-MB, the serum concentrations of these markers among individuals with spontaneous ischaemia were compared with those of patients without ischaemia and with the reference limits found in apparent healthy noncardiac subjects. Furthermore, in order to reduce the large differences between the levels of S-myoglobin, S-CK and S-CK-MB of the patients, the data were normalized. The normalization within the individual patient was performed by dividing each value by the mean of all eight measurements obtained during each period. As a reference interval for these fractional concentrations the 95% confidence interval (mean ± 2 pooled SD) of the six patients without episodes of ischaemia during period A (Table 2) was calculated.
Results ST-SEGMENT MONITORING AND EXERCISE TESTING
All patients had a positive exercise test with significant ST-depression (mean: 2-8 mm, range: 1-7 mm) and the mean maximum workload was 1120W (range: 80-170) (Table 1). Table 2 shows the distribution of spontaneous ischaemic episodes during periods A and B. During period A, a total of 47 episodes (all silent) were noted in four patients. The total duration for all episodes of all patients was 599 min (range 37-336). Monitoring in period B detected 47 episodes of ischaemia obtained in
1(28) — — 1(11) — — 6(74)
Total 10(172) 4(54) 1(37) 32(336) 4(106) 2(21) 2(38) 5(149) 1(27) 3(69) 30(394)
seven patients and 91% were asymptomatic. The total duration of ischaemia for all episodes of all patients was 804 min (range 21-394). MYOGLOBIN
The median concentration and inter quartile range of Smyoglobin for each sampling time of periods A and B are shown in Fig. 1 (upper panel). During both periods, Smyoglobin had a significant circadian variation, with highest values in the morning and lowest values in the late afternoon (P005). The fractional concentration of Smyoglobin in the patients with spontaneous ischaemia during period A (No 1,2,3,10) and period B (1,2, 3, 5, 8, 9, 10) was largely within the reference interval (Fig. 3: upper panel). CK
In Fig. 1 (middle panel), the median activity and inter quartile range of S-CK during periods A and B is displayed. During both periods a minor but significant variation in S-CK was found during the day and with highest activity at 1900 h (P = 0021). No difference in S-CK level was found between period B (after exercise testing) and period A (P = 0-084). The median and inter quartile ranges in S-CK for patients with and without ischaemia during period A are shown in Fig. 2 (middle panel). The values of the patients with ischaemic attacks did not exceed the upper reference limit of non-cardiac patients, and no difference between patients with and without ischaemia were found (P>005). During period B, two patients (Nos 1 and 2) had minor peak values exceeding the upper 95% reference interval of the fractional
No cell damage after myocardial ischaemia? 191
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Figure 1 The median and inter quartile range of S-myoglobin (upper panel) S-CK (middle panel) and S-CK-MB (lower panel) in 10 patients with stable angina pectoris at eight sampling times during 24 h at minimal activity (• = period A) and during 24 h following exercise at 0815 h ( • = period B).
activities (Fig. 3: middle panel) at 1200 h, but these peaks had no relation to the occurrence of ischaemic episodes.
I
Figure 2 The median and inter quartile range of S-myoglobin (upper panel) S-CK (middle panel) and S-CK-MB (lower panel) in four patients with and six patients without spontaneous ischaemia during period A. The upper reference limit (dotted line) for apparently healthy non-cardiac subjects is indicated for S-myoglobin and S-CK-MB. For S-CK this limit is 270 U 11 '. * = ischaemia; D = n o ischaemia.
S-CK-MB exceeding the upper reference limit of healthy subjects, but his S-CK-MB curve showed no changes Fig. 1 (lower panel) displays the median concentration throughout the day compatible with myocardial release of and inter quartile range of S-CK-MB during periods A the enzyme (Fig. 2: lower panel); this patient had a correand B. No circadian variation was found during the two spondingly high level of CK-MB during period B. At each periods (P> 0-45), and the level of period B (after exercise separate sampling time no difference between the S-CKtesting) was not higher than the level of j>eriod A (P = MB values of patients with and without ischaemia could 0-69). During period A, one patient (no. 3) had values of be found (P>005) (Fig. 2: lower panel). During both CK-MB
192 K. Nerregaard-Hansen etal.
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Figure 3 Fractional concentration of myoglobin (upper panel), CK (middle panel) and CK-M B (lower panel) in four patients with spontaneous ischaemic attacks during period A(V — V) (Nos 1,2,3,10) and in seven patients with spontaneous attacks during period B (C C) (Nos 1, 2, 3, 5, 8, 9, 10). The shaded area represents the 95% reference interval (mean ±2 pooled SD) calculated from the six patients without spontaneous ischaemia during period A.
periods the fractional concentration of S-CK-MB of patients with spontaneous ischaemia was largely within the reference interval (Fig. 3: lower panel). Discussion
This study shows that in patients with severe stable angina no detectable amount of myoglobin, CK and CK-
MB is released into the peripheral circulation in relation to prolonged and repetitive spontaneous ischaemic episodes or exercise-induced ischaemia. In several animal studies periods of coronary occlusion above 20-30 min have resulted in myocardial cell necrosis with release of myocardial enzymes, and briefer if repetitive (5-15 min) episodes may also lead to myocardial necrosis'10'. The total ischaemic burden observed in some of the patients in our study exceeds the ischaemic burden provoked in these animal studies'10'. In these experiments, however, ischaemia was induced by total occlusion of one of the coronary arteries, whereas ischaemia in stable angina is normally related to a partial reduction of coronary blood flow and to increased myocardial oxygen demands, but not to coronary occlusion. Thus, Block et al. (1983) found that a 50% reduction in left main coronary artery diameter did not lead to myocardial necrosis or release of myoglobin'21'. Furthermore the collateral circulation which is often present in patients with chronic stable angina may partly protect ischaemic myocardium'22'. Assessment by measurement in peripheral blood samples of increased release of intracellular proteins from myocardial cells instead of samples from the coronary sinus may be less sensitive, because of a dilution effect'23'. Sylven et al. (1984) measured myoglobin in the coronary sinus blood after paced-induced angina and they suggested a minor release of myoglobin into the coronary circulation124'. Blood sampling from the coronary sinus however would not be possible over long study periods as in this study. To compensate for the dilution effect we have used highly sensitive analytical methods. For the analyses of CK-MB a recently developed immunoassay with low imprecision and low detection limit was used1'71. The immunoinhibition method most often used for CK isoenzyme analysis in patients suspected of AMI is not suitable because of high imprecision in the low range1'1. Clinical studies have documented that release of intracardial proteins, such as myoglobin, CK and CK-M B into the circulation is a sensitive indicator of myocardial necrosis in patients with AMI'1"3'. Minor, but significant release has also been shown to take place in some patients with unstable angina pectoris'451. It is most likely that this release reflects myocardial cell death and not leakage from reversibly damaged cells. Subendocardial necrosis in unstable angina patients has indeed been proved by other methods such as technetium-99m pyrophosphate myocardial scintigraphy125'. The fact that no increased release of myoglobin, CK and CK-MB could be detected in our patients therefore seems to exclude myocardial necrosis. Ourfindingsare in accordance with those of Baadsgaard and Schmidt (1984) and Marmor et al. (1978) who found no release of myoglobin and creatine kinase isoenzymes in patients with stable angina pectoris after exercise testing"-81. Neither did Bagger et al. (1982) find release followed by pacing induced ischaemia'23'. In conclusion prolonged and repetitive spontaneous symptomatic or asymptomatic ischaemia and exerciseinduced ischaemia in stable angina pectoris are not associated with irreversible myocardial cell damage as measured from release of myoglobin, CK or CK-MB.
No cell damage after myocardial ischaemia? 193
This study was kindly supported by a grant from 'Guldsmed AL and D. Rasmussensmindelegat'.
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