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(Circulation. 1996;94:2373-2380.)
© 1996 American Heart Association, Inc.

Articles

Plasma Protein Acute-Phase Response in Unstable Angina Is Not Induced by Ischemic Injury

Giovanna Liuzzo, MD; Luigi M. Biasucci, MD; Antonio G. Rebuzzi, MD; J. Ruth Gallimore, BSc; Giuseppina Caligiuri, MD; Gaetano A. Lanza, MD; Gaetano Quaranta, MD; Claudia Monaco, MD; Mark B. Pepys, MD; Attilio Maseri, MD

Istituto di Cardiologia, Universita' Cattolica del S. Cuore, Rome, Italy (G.L., L.M.B., A.G.R. G.C., G.A.L., G.Q., C.M., A.M.), and Immunological Medicine Unit, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK (J.R.G., M.B.P.).

Correspondence to Giovanna Liuzzo, MD, Istituto di Cardiologia, Universita' Cattolica del Sacro Cuore, Largo A. Gemelli, 8, 00168 Rome, Italy.

	Abstract

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Background Elevated levels of C-reactive protein (CRP) are associated with an unfavorable clinical outcome in patients with unstable angina. To determine whether ischemia-reperfusion injury causes this acute-phase response, we studied the temporal relation between plasma levels of CRP and ischemic episodes in 48 patients with unstable angina and 20 control patients with active variant angina, in which severe myocardial ischemia is caused by occlusive coronary artery spasm.

Methods and Results Blood samples were taken on admission and subsequently at 24, 48, 72, and 96 hours. All patients underwent Holter monitoring for the first 24 hours and remained in the coronary care unit under ECG monitoring until completion of the study. On admission, CRP was significantly higher in unstable angina than in variant angina patients (P<.001). In unstable angina, 70 ischemic episodes (1.5±2 per patient) and in variant angina 192 ischemic episodes (9.6±10.7 per patient) were observed during Holter monitoring (P<.001), for a total ischemic burden of 14.8±30.2 and 44.4±57.2 minutes per patient, respectively (P<.001). The plasma concentration of CRP did not increase in either group during the 96 hours of study, even in patients who had episodes of ischemia lasting >10 minutes.

Conclusions The normal levels of CRP in variant angina, despite a significantly larger number of ischemic episodes and greater total ischemic burden, and the failure of CRP values to increase in unstable angina indicate that transient myocardial ischemia, within the range of duration observed, does not itself stimulate an appreciable acute-phase response.

Key Words: angina • ischemia • inflammation • reperfusion

	Introduction

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Unstable angina is a complex syndrome, the pathophysiological mechanisms of which are still incompletely understood. The recent observation of inflammatory infiltrates in unstable coronary plaques^{1 2 3 4 5 6} suggests that inflammatory processes may contribute to the pathogenesis of this syndrome by stimulating or enhancing local hemostatic and vasoconstrictor responses. These postmortem observations are confirmed by clinical studies demonstrating activated circulating neutrophils,^{7 8 9} lymphocytes,¹⁰ and monocytes^{11 12} and increased concentrations of the highly sensitive acute-phase reactant C-reactive protein (CRP)^{13 14} in patients with unstable angina. We have recently confirmed the frequent elevation of acute-phase protein concentrations in severe unstable angina and demonstrated the unfavorable short-term prognostic significance of raised levels of CRP,¹⁵ which were unrelated to myocardial cell necrosis, as creatine kinase and troponin T levels were within normal limits.¹⁵ However, many experimental studies have shown that short periods of ischemia and reperfusion are a powerful proinflammatory stimulus, capable of inducing leukocyte and complement activation, cytokine production, and acute-phase protein synthesis.^{16 17 18 19 20 21 22 23 24 25 26} The acute-phase response in severe unstable angina, with its remarkable prognostic value, may therefore simply be the biochemical marker of recurrent and prolonged myocardial ischemic episodes with consequent ischemia-reperfusion injury.

To elucidate this question, we compared the time course of plasma levels of CRP in relation to ischemic episodes in patients with unstable angina with that in patients with variant angina, a human model of transmural myocardial ischemia caused by occlusive epicardial coronary artery spasm.²⁷ Our results demonstrate that CRP levels were not increased in patients with active variant angina despite a significantly higher number of ischemic episodes and a greater total ischemic burden during Holter monitoring. Furthermore, CRP levels in unstable angina patients were also unrelated to the number and duration of ischemic episodes. Thus, transient myocardial ischemia by itself does not appear to stimulate the significant acute-phase response commonly observed in unstable angina.

	Methods

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Patient Population
We prospectively studied two groups of patients. Group 1 initially comprised 54 of the 115 patients admitted to our coronary care unit between March 1993 and July 1994 with severe unstable angina (45 men and 9 women; mean age [SD], 61.5 [8.4] years). Inclusion criteria were angina at rest with 2 episodes in the last 48 hours or 1 episode lasting >20 minutes, ST-segment shift diagnostic for myocardial ischemia during angina attacks, the last episode of chest pain in the last 24 hours, and no elevation in serum creatine kinase, lactate dehydrogenase, and troponin T on admission. Symptoms of unstable angina started 2 to 30 days, mean (SD) 13 (10), before admission. Exclusion criteria were absence of ischemic episodes during the last 24 hours before hospitalization (29 patients); myocardial infarction within the previous month (10 patients); elevated creatine kinase and/or lactate dehydrogenase on admission (9 patients); valvular heart disease (1 patients); left bundle branch block complicating ST-segment analysis (3 patients); and intercurrent inflammatory or neoplastic conditions likely to be associated with an acute-phase response (9 patients). At the end of the study, when troponin T levels were measured, a further 6 patients were excluded, 2 individuals because they had increased values on admission (range, 0.23 to 0.41 µg/L) and 4 cases who developed a slight increase of troponin T after 48 to 72 hours of study (range, 0.21 to 0.47 µg/L). Thus, group 1 finally consisted of 48 patients with unstable angina and no detectable signs of myocardial cell necrosis (troponin T values <0.1 µg/L). All patients underwent ECG Holter monitoring for the first 24 hours and received aspirin, calcium channel blockers, and/or oral nitrates and/or ß-blockers. Intravenous nitrates and heparin were given as required by recurrent ischemic episodes. Coronary angiography was performed in 43 of 48 patients as required by clinical criteria.

Group 2 comprised 20 of the 29 patients hospitalized in our coronary care unit between March 1993 and July 1994 with active variant angina (15 men and 5 women; mean age [SD], 56 [11] years). The diagnosis of variant angina was based on a typical history of recurrent angina attacks occurring unpredictably and predominantly at rest, usually in the early morning, lasting a few minutes, and promptly responding to sublingual nitrates. The diagnosis was confirmed by ECG alteration during angina attacks, with transient episodes of ST-segment elevation, by the positive results of hyperventilation or ergonovine provocation tests, and by spontaneous or induced spasm during coronary angiography. Exclusion criteria were negative results of provocation testing (5 patients) and intercurrent inflammatory conditions likely to be associated with an acute-phase response (4 patients). After the assessment of diagnosis, all patients underwent Holter monitoring for the first 24 hours and received aspirin; sublingual nitrates were given in case of ischemia. No oral or intravenous nitrates, calcium channel blockers, or ß-blockers were given during the 24 hours of monitoring; subsequently, group 2 patients were treated with high doses of calcium channel blockers, oral nitrates, and aspirin.

A venous line was inserted in both groups of patients for infusion of nitrates and heparin as required in group 1 and for infusion of saline in group 2. All patients in both groups remained in the coronary care unit under ECG monitoring of the lead with the most striking ischemic ST changes until completion of the study.

Study Protocol
Clinical History
The number and the duration of angina attacks during the 48 hours before admission were carefully investigated because CRP levels begin to rise 6 hours after an acute stimulus and peak after 24 to 48 hours.^{28 29}

Blood Sampling
Venous blood was taken in all patients on admission to the hospital and subsequently at 24, 48, 72, and 96 hours. Additional samples were taken 6, 24, 48, and 72 hours after one ischemic episode lasting >10 minutes in 9 patients with variant angina. Finally, in 15 patients with variant angina, an additional sample was taken after 16±4 days. Coded plasma samples were stored at -70°C and analyzed for CRP and troponin T in a single batch at the end of the study. All clinical classifications and therapeutic decisions were thus independent of these results. Total creatine kinase was determined routinely at the time of sampling in group 1 patients.

ECG Holter Monitoring
ECG Holter monitoring was performed in the first 24 hours in all patients with the use of 2-channel Oxford Medilog MR-45 tape recorders, which perform real-time analysis of the ST segment. Bipolar electrodes were applied to record CM5 lead on channel 1 and the lead most similar to that showing maximal ST-segment change on standard ECG (CM2 or CM3 for anterior ST change, and modified aVF for inferior ST change) on channel 2. The recordings were analyzed with an Oxford Excel 2.0 device. An ischemic episode was defined as transient rectilinear or downsloping ST-segment depression 0.1 mV (1 mm) and/or as transient ST-segment elevation 0.1 mV (1 mm), measured 0.08 second after the J point, lasting at least 1 minute. The duration of ischemic episodes was calculated from the onset of ST alteration to return to baseline. An isoelectric segment lasting at least 1 minute was required between episodes. The variables evaluated included the number of ischemic episodes, the average duration of ischemia, the duration of the longest ischemic episode, and the total ischemic burden (sum of duration in minutes of all the ischemic episodes recorded during the 24 hours for each patient). Other variables analyzed were the type of ischemic changes (ST-segment depression or elevation), the maximum ST-segment depression or elevation during the episodes, and the ECG lead showing the ST-segment alteration. These variables were carefully recorded and reviewed by an expert cardiologist who was unaware of the clinical and analytic data.

In-Hospital Follow-up
The number and length of recurrent ischemic episodes during the subsequent 96 hours of study were also carefully recorded.

Provocation Tests
The clinical diagnosis was confirmed in all group 2 patients by provocation tests. In 8 of 20 individuals, the hyperventilation test was performed.³⁰ In the remaining 12 patients, the ergonovine test was performed by intravenous injection of incremental doses of ergonovine maleate.^{31 32} A standard 12-lead ECG was continuously recorded and blood pressure (cuff) obtained at regular intervals throughout each test and for 10 minutes thereafter. The tests were considered positive when >0.1 mV of ST elevation occurred in at least 2 leads.

Coronary Angiography
Coronary angiography was performed in 43 of 48 group 1 patients: in 22 with refractory angina necessitating urgent revascularization it was performed within 7 days; in 15 it was performed 7 to 15 days after admission; and in 6 others it was performed 7 to 15 days after they had suffered myocardial infarction. One patient died before coronary angiography, and in 4 group 1 patients, the procedure was not performed because symptoms improved.

Coronary angiography was performed in all group 2 patients. To confirm the diagnosis of variant angina, intracoronary injection of incremental doses of ergonovine maleate (4 to 32 µg, 6 patients)³³ or serotonin (10^-7 to 10^-5 mol/L, 5 patients)³⁴ was performed in 11 patients without significant coronary artery disease (coronary stenosis <50%). The artery was chosen on the basis of previous regional ECG localization of myocardial ischemia during chest pain; ECG and blood pressure were continuously monitored. Four patients spontaneously developed focal coronary spasm, and in the remaining 5 patients we did not perform provocation tests during coronary angiography because they had severe stenosis of the coronary artery involved.

Ethics and Consent
The study was approved by the Ethics Committee of the Catholic University of Rome; all patients gave their informed consent.

C-Reactive Protein and Troponin T Assays
CRP was assayed by automated monoclonal antibody solid-phase sandwich-type enzyme immunoassay on the Abbott Im_x instrument calibrated with the World Health Organization's International Reference Standard for CRP Immunoassay (85/506). Calibrators and controls were prepared from isolated pure CRP.³⁵ The range of values detected by the assay is 0.005 to 3.0 mg/dL, with intra-assay and interassay coefficients of variation of <10% throughout. The median normal value for CRP is 0.08 mg/dL, with 90% of normal values <0.3 and 99% <1.0 mg/dL.³⁶ Plasma levels of troponin T were measured by enzyme immunoassay (Boehringer); normal value was <0.1 µg/L.

Statistics
As CRP values, the number and the duration of ischemic episodes do not follow a normal distribution, the Mann-Whitney test was used for comparisons between groups. Comparisons within groups were carried out with the use of the Friedman test; for values of P<.05, pairwise comparisons were carried with the use of the Wilcoxon test with the Bonferroni correction. Correlations were determined using Spearman's rank correlation test. The remaining continuous variables were compared using t tests for paired and unpaired variables as appropriate. Proportions were compared with the use of the ² test. CRP values are expressed as median and range; the remaining variables are expressed as mean±SD. Values of P<.05 (two-tailed) were considered statistically significant.

	Results

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Clinical Characteristics and Provocation Tests
The clinical characteristics and angiographic findings in both groups of patients are summarized in Table 1.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics and Angiographic Findings in Unstable and Variant Angina Patients

During hyperventilation or ergonovine provocation tests, all group 2 patients had anginal pain, which was associated with marked ST-segment elevation. This myocardial ischemia was completely reversed within 2 to 10 minutes by intravenous isosorbide dinitrate.

In 11 group 2 patients without significant coronary artery disease, intracoronary injection of ergonovine maleate (6 patients) or serotonin (5) was performed, and all of them developed focal occlusive or subocclusive coronary spasm (4 of left anterior descending, 5 of right and 2 of left circumflex coronary artery). Another 4 patients (2 with angiographically normal coronary arteries and 2 with significant coronary stenosis) spontaneously developed focal coronary spasm (3 of the left anterior descending and 1 of the right coronary artery). In all patients, coronary spasm was promptly relieved by 1 to 2 mg of intracoronary isosorbide dinitrate.

Angina Before Coronary Care Unit Admission
During the 48 hours before coronary care unit admission, all group 1 and group 2 patients had experienced at least 2 episodes of angina or 1 episode lasting >20 minutes. The total number of angina attacks reported was 144 in group 1 (3±2 per patient; range, 1 to 10) and 106 in group 2 (5.3±4 per patient; range, 2 to 15) (P<.001). The longest angina episode per patient ranged from 2 to 40 minutes in group 1 (median, 10 minutes; in 12 patients the longest episode lasted >10 minutes and in 5 it lasted >20 minutes) and in group 2 the range was 1 to 15 minutes (median, 5 minutes; in 2 patients the longest episodes lasted >10 minutes) (P<.001) (Table 2).

View this table: [in this window] [in a new window]   Table 2. Ischemic Episodes in Unstable Angina and Variant Angina Patients

ECG Holter Monitoring
During the 24 hours of Holter monitoring, 29 of 48 (60%) group 1 patients and 18 of 20 (90%) group 2 patients had at least one ischemic episode (P=.034). The total number of ischemic episodes and the total ischemic burden were both significantly greater in group 2 than in group 1, but the mean duration of episodes was longer and the number of patients with all episodes lasting <5 minutes was smaller in group 1 (Table 2).

Ischemic Episodes After the First 24 Hours of Study
After the first 24 hours, a similar proportion 38 of 48 (79%) of group 1 patients and 17 of 20 (85%) of group 2 patients had 1 ischemic episode (P=.83). The number per patient was higher in group 2 but the duration of the longest ischemic episode per patient was slightly greater in group 1 (Table 2). During the 96 hours of study, 9 group 2 patients (8 patients without significant coronary artery disease and 1 with stenosis of the left anterior descending coronary artery) had episodes of angina lasting >10 minutes (16±4.2 minutes; range, 12 to 25). In all episodes, ST-segment shifts diagnostic of transmural myocardial ischemia were observed on the 12-lead ECG. Four patients had ST-segment elevation in the anterior leads, 4 in the inferior leads, and 1 in the lateral leads. The ischemia was relieved by intravenous injection of isosorbide dinitrate plus calcium channel blockers in 2 cases.

C-Reactive Protein Concentrations
At the time of hospital admission, CRP concentration was significantly higher in group 1 (median, 0.61 mg/dL; range, 0.06 to 8.25 mg/dL) than in group 2 patients (median, 0.21 mg/dL; range, 0.03 to 0.6 mg/dL) (P<.001). Elevated CRP (>0.3 mg/dL) was observed in 35 of 48 (73%) group 1 patients and in 4 of 20 (20%) group 2 patients (P<.001) (Fig 1). The values on admission were unrelated to the number and duration of preceding angina episodes (see below).

View larger version (13K): [in this window] [in a new window]   Figure 1. C-reactive protein (CRP) concentration in individual patients. Left, unstable angina (); right, variant angina (). CRP is <0.3 mg/dL in 90% of healthy subjects, shown by broken line.

During the subsequent 96 hours of study, the plasma concentration of CRP did not increase significantly in group 1 patients, changing from an entry value of 0.61 (0.06 to 8.25) mg/dL to 0.64 (0.06 to 8.25) mg/dL at 24 hours, 0.78 (0.06 to 8.77) mg/dL at 48 hours, 0.76 (0.06 to 12.6) mg/dL at 72 hours, and 0.70 (0.08 to 9.89) mg/dL at 96 hours (Fig 2). In 17 of 48 (35%) group 1 patients, the highest value was observed at entry, in 8 (17%) the peak value was observed at 24 hours, in 10 (21%) at 48 hours, in 8 (17%) at 72 hours, and in 5 (10%) at 96 hours. In 17 of 48 group 1 patients (35%), the peak value of CRP was observed within 24 hours from admission, although they continued to have ischemic episodes (2.9±1.6 per patient) throughout the study.

View larger version (10K): [in this window] [in a new window]   Figure 2. Changes in median levels of C-reactive protein (CRP) after admission in unstable angina () and variant angina () patients. During the 96 hours of study, the plasma concentration of CRP did not increase in either group of patients (Friedman test: P=.37 and P=.062, respectively).

In group 2 patients, CRP changed from an entry value of 0.21 (0.03 to 0.6) mg/dL to 0.11 (0.03 to 0.54) mg/dL at 24 hours, 0.1 (0.02 to 0.43) mg/dL at 48 hours, 0.09 (0.03 to 0.47) mg/dL at 72 hours, and 0.09 (0.03 to 0.29) mg/dL at 96 hours (Fig 2). In 3 of 20 (15%) group 2 patients (2 with normal values and 1 with a high value at entry), a slight increase of CRP was observed during the study (entry CRP values, 0.18 to 0.23 to 0.37 mg/dL; peak values, 0.47 to 0.44 to 0.44 mg/dL, respectively). In 3 of 20 (15%) patients with high values at entry, CRP levels decreased to <0.3 mg/dL; in the remaining 14 of 20 (70%) group 2 patients, CRP levels remained within the normal range in all samples. A delayed increase in CRP was also excluded, as CRP levels 16±4 days after admission were normal in the 15 group 2 patients in whom the assessment was performed (median, 0.09; range, 0.05 to 0.28; P=.78 versus 96 hours). Moreover, in the 9 group 2 patients who experienced episodes of transmural ischemia lasting >10 minutes, CRP levels 6, 24, 48, and 72 hours after the event did not increase compared with the entry value (Fig 3).

View larger version (17K): [in this window] [in a new window]   Figure 3. Changes in C-reactive protein (CRP) levels after ischemic episodes lasting >10 minutes in 9 variant angina patients (, median values). During the 72 hours after the ischemic episodes studied, the plasma concentration of CRP did not increase compared with the entry value (Friedman test: P=.76).

There was no correlation in either group between the value of CRP at entry and the time from the last episode of angina (unstable angina: r=-.1, P=.59; variant angina: r=.22, P=.22) or its duration (unstable angina: r=.03, P=.86; variant angina: r=-.13, P=.79). There was also no correlation between the values of CRP at entry and the total number of episodes of angina or the duration of the longest episode of angina during the 48 hours before admission (Fig 4).

View larger version (18K): [in this window] [in a new window]   Figure 4. Correlation between the entry values of C-reactive protein (CRP) and the total number of angina episodes (top) or the duration of the longest angina episode (bottom) recorded during the 48 hours before admission. Left, unstable angina (); right, variant angina (). CRP values on admission were unrelated (Spearman's rank correlation test) to the number of preceding ischemic episodes (unstable angina: r=.2, P=.17; variant angina: r=.15, P=.53) or their duration (unstable angina: r=-.1, P=.5; variant angina: r=-.12, P=.61).

Furthermore, in neither group were there any significant correlations between the number of ischemic episodes, the total ischemic burden, or the duration of the longest ischemic episodes during the first 24 hours and the CRP values observed after 24, 48, and 72 hours (Figs 5, 6, and 7).

View larger version (18K): [in this window] [in a new window]   Figure 5. Correlation between the values of C-reactive protein (CRP) at 24 hours and the number of ischemic episodes (top) or the total ischemic burden (bottom) recorded by ECG Holter monitoring during the first 24 hours. Left, unstable angina (); right, variant angina (). CRP values at 24 hours were unrelated (Spearman's rank correlation test) to the number of ischemic episodes (unstable angina: r=-.076, P=.62; variant angina: r=.09, P=.69) or the total ischemic burden (unstable angina: r=-.079, P=.6; variant angina: r=.06, P=.8).

View larger version (17K): [in this window] [in a new window]   Figure 6. Correlation between the values of C-reactive protein (CRP) at 48 hours and the number of ischemic episodes (top) or the total ischemic burden (bottom) recorded by ECG Holter monitoring during the first 24 hours. Left, unstable angina (); right, variant angina (). CRP values at 48 hours were unrelated (Spearman's rank correlation test) to the number of ischemic episodes (unstable angina: r=.024, P=.88; variant angina: r=-.06, P=.95) or the total ischemic burden (unstable angina: r=.03, P=.84; variant angina: r=-.02, P=.98).

View larger version (17K): [in this window] [in a new window]   Figure 7. Correlation between the values of C-reactive protein (CRP) at 72 hours and the number of ischemic episodes (top) or the total ischemic burden (bottom) recorded by ECG Holter monitoring during the first 24 hours. Left, unstable angina (); right, variant angina (). CRP values at 72 hours were unrelated (Spearman's rank correlation test) to the number of ischemic episodes (unstable angina: r=.12, P=.47; variant angina: r=.07, P=.7) or the total ischemic burden (unstable angina: r=.17, P=.31; variant angina: r=-.01, P=.9).

Finally, patients in both groups were subgrouped according to the duration of the ischemic episodes: those with only Holter-recorded ischemic episodes lasting <5 minutes (8 patients with unstable angina and 7 with variant angina) and those with at least one Holter-recorded ischemic episode lasting >10 minutes (18 cases with unstable angina and 10 with variant angina). During the 96 hours of study, the plasma concentration of CRP did not change even in patients with the long-lasting ischemic episodes (Fig 8).

View larger version (11K): [in this window] [in a new window]   Figure 8. Changes in median levels of C-reactive protein (CRP) after admission in patients of both groups subgrouped according to the duration of their ischemic episodes. Patients with Holter-recorded ischemic episodes lasting <5 minutes (8 patients with unstable angina, ; 7 patients with variant angina, ); patients with Holter-recorded ischemic episodes lasting >10 minutes (18 patients with unstable angina, ; 10 patients with variant angina, ). During the 96 hours of study, the plasma concentration of CRP did not change even in patients with long-lasting ischemic episodes (Friedman test: unstable angina, P=.89; variant angina, P=.06).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our study demonstrates that relatively short episodes of ischemia-reperfusion, such as those commonly observed in patients with unstable angina and variant angina, do not themselves stimulate a significant acute-phase response. Variant angina is a valuable human model of experimental ischemia-reperfusion characterized by frequent episodes of transmural ischemia, caused by occlusive epicardial coronary artery spasm, and its pathogenesis clearly differs from that of unstable angina.²⁷

Increased concentrations of CRP, a sensitive marker of inflammation, have been reported in patients with unstable angina,^{13 14} but in these studies myocardial necrosis, an established cause of the acute-phase response, was not excluded. We have recently confirmed the frequent elevation of acute-phase proteins in unstable angina and demonstrated that it was unrelated to myocardial cell necrosis, as creatine kinase and troponin T levels were within normal limits.¹⁵ However, in that study we could not establish whether this acute-phase response was related to recurrent and prolonged myocardial ischemic episodes with consequent ischemia-reperfusion injury or to inflammatory stimuli arising in the coronary tree and/or elsewhere.

Experimental studies have shown that periods of ischemia as short as 15 minutes followed by reperfusion elicit a cascade of proinflammatory reactions that include production of oxygen-derived free radicals,¹⁹ activation of the complement system,²² adherence of neutrophils to the coronary endothelium,^{37 38} leukocyte-mediated injury of the myocardial cells,³⁹ and production of cytokines,^{23 24 25 26} including interleukin (IL)-6 and IL-1, which are the major determinants of acute-phase protein production.^{40 41} In patients, neutrophil activation with signs of endothelial injury and release of proinflammatory cytokines have been demonstrated in acute myocardial infarction^{21 42 43 44} and after coronary angioplasty.^{45 46} Furthermore, in unstable angina patients a significantly increased urinary concentration of leukotriene E₄ was observed immediately after ischemia compared with 2 days later.⁴⁷ A recent report of elevated plasma levels of soluble P-selectin after angina attacks in patients with unstable angina compared with stable effort angina and control subjects, contained no information on the number and duration of the ischemic episodes.⁴⁸ Plasma levels of soluble P-selectin have also been reported to increase in the coronary circulation after acute myocardial ischemia induced by coronary spasm.⁴⁹

Plasma levels of CRP start to rise about 6 hours after an acute stimulus, reaching a peak within about 48 hours and, with abrupt cessation of the stimulus, values then decrease exponentially at a rate close to the measured plasma half-life of CRP of about 19 hours.^{28 29} Thus, if ischemia-reperfusion does trigger an acute-phase response, the peak values of CRP should be observed at 48 to 72 hours in patients with ischemic episodes before admission or during the first 24 hours, and there should be a persistent increase in CRP in patients with crescendo or refractory angina. However, in the present study frequent episodes of transmural ischemia in active variant angina were not associated with increased CRP production. Only 4 of 20 (20%) patients with variant angina had CRP >0.3 mg/dL on admission, a proportion close to the 13% of patients with elevated CRP observed in chronic stable angina patients during symptom-free periods.^{13 15} Despite 192 ischemic episodes during 24 hours of ECG Holter monitoring in variant angina patients and 95 ischemic episodes during the subsequent 72 hours of study, only 2 of the 16 patients with normal CRP values at entry showed a rise in CRP >0.3 mg/dL, and only 1 of the 4 patients with elevated levels of CRP at entry had a further slight increase while CRP values actually fell in the other 3. Although the majority of the ischemic episodes in variant angina were brief, 10 of 20 (50%) patients experienced at least 1 episode of transmural myocardial ischemia >10 minutes, comparable to the mean duration of the ischemic episodes in the unstable angina group.

In unstable angina patients, although we observed 70 ischemic episodes during the 24 hours of ECG Holter monitoring and 119 ischemic episodes during the subsequent 72 hours of study, 35% of patients had their highest CRP value on admission, and the rise in CRP concentration after admission observed in the others was unrelated to the number and duration of ischemic events in the preceding 48 hours. These findings strongly suggest that episodes of ischemia-reperfusion are not by themselves a sufficient cause of the acute phase response in unstable angina.

The failure of current optimal therapy to reduce levels of CRP during the study in unstable angina patients confirms our previous observation¹⁵ and may suggest that an underlying inflammatory process contributes to the pathogenesis of unstable angina.

Conclusions
The normal levels of CRP in variant angina, despite significantly larger number of ischemic episodes and greater total ischemic burden during Holter monitoring, and the lack of relation between the number and duration of ischemic episodes and CRP levels in unstable angina suggest that transient myocardial ischemia, within the range of duration observed, does not itself stimulate an appreciable acute-phase response. Thus, the increased production of sensitive acute-phase proteins in unstable angina is likely to reflect inflammatory activity within the coronary arteries and/or elsewhere in the body.

	Acknowledgments

 
This study was supported by National Research Council (CNR)-Targeted Project "Prevention and Control Disease Factors," Rome, Italy (research grant 94.00518.PF41), MRC Programme Grant G7900510 to Dr Pepys, and in part by the European Community (Biomed 2 research grant PL951505). We are indebted to the nurses of the Coronary Care Unit at Policlinico Gemelli for their assistance.

Received December 28, 1995; revision received June 11, 1996; accepted June 13, 1996.

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