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Plasma Oxidase Assay for Screening of Myocardial Infarction
Plasma Oxidase Assay for Screening of Myocardial Infarction GABRIEL PEREZ LASALA, MO, TIMOTHY WRIGHT, MO, KHIOIR OSMAN, MO, SAMER SIOUFFI, MO, THOMAS N. SKELTON, MO, PATRICK H. LEHAN, MO, ANGEL K. MARKOV, MO
ABSTRACT: The availability of techniques such as surgical reperfusion, angioplasty, and thrombolysis for the treatment of acute myocardial infarction (AMI) has revived interest in seeking an early detectable biochemical marker diagnostic for AMI. Therefore, we investigated whether an unidentified oxidase that is released by activated neutrophils at the onset of AMI could be used as an early diagnostic assay. The conversion by plasma oxidase of 1 ~M of adrenaline to 1 ~M of adrenochrome represents the plasma oxidase activity (POA) of 1 UIL. Fifty patients suspected of having AMI, 40% of whose electrocardiograms were nondiagnostic for AMI, were admitted to the coronary care unit, and venous blood samples were obtained for determination of the POA and creatine phosphokinaseMB levels. Healthy volunteers (n = 12) served as control subjects, and 8 patients with pneumonia whose leukocyte counts were greater than 15,000 ~L were included in the study. In those with AMI (n = 22), as determined by serial creatine phosphokinase-MB, the mean POA (± standard error of the mean) was 233 ± 13 UIL, and in those with angina and no AMI (n = 28) was 127 ± 5 UIL (P < 0.0001). In the control group, mean POA (± standard error of the mean) was 84 ± 5 UIL (control versus angina; P < 0.01) and for those with infection was 214 ± 10 UIL. At admission, the creatine phosphokinase-MB was diagnostic for only 12 of the 22 patients with AMI (sensitivity rate of 54%), whereas in 21 of those patients, the POA values were diagnostic for AMI (sensitivity rate of95%). Determination of POA may represent an alternative sensitive From the University of Mississippi Medical Center, Jackson, Mississippi. Presented at the 45th Annual Meeting of the Southern Society for ClinicalInvestigation, New Orleans, Louisiana, January 30-February 1, 1991 (Clin Res 1991; 39[4]:799A). Correspondence: Angel K. Markov, MD, Department of Medicine, Division of Cardiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
method to screen for AMI or ischemia in patients with anginal symptoms and no evidence of acute infection. KEY INDEXING TERMS: Myocardial infarction; Oxidase; Assay; Screening. [Am J Med Sci 1994;308(3):157-161.]
P
olymorphonuclear leukocyte (PMN)-deriyed oxyradicals have been shown to contribute to the tissue injury associated with acute myocardial infarction (AMI).l In addition, activated PMNs release other potent substances that likely have deleterious effects (proteases, leukotrienes, thromboxane, and others). In inflammatory conditions in which PMN tissue infiltration occurs, there is evidence that the activated PMN can oxidize adrenaline to adrenochrome. 2 - s Medium isolated after stimulation of the PMN retained its ability to oxidize adrenaline to adrenochrome. 2 - 4 Plasma from patients with inflammatory diseases causes more oxidation of adrenaline to adrenochrome than do the sera from healthy volunteers. 2 - 4 Matthews et al2-4 demonstrated the presence of an adrenaline oxidase in patients with acute inflammatory conditions as a biochemical marker for plasma oxidase activity (POA). Although the role of the oxidase is not defined, it may act as a natural defense mechanism against endogenously released adrenaline in the course of myocardial infarction (MI), thereby diminishing the likelihood for occurrence of ventricular arrhythmias. Creatinine phosphokinase (CPK) consists of 2 subunits, M and B, which form 3 isoenzymes denoted as CPK-MM, CPK-BB, and CPK-MB. The latter isoenzyme usually is found in the cardiac muscle, but it could be detected in small quantities in other tissue. Although CPK-MB has no absolute specificity rate, an elevated level in serum generally is accepted as the best indicator for AMI. The sensitivity and specificity rates by serial CPK-MB sampling approach 90% 5-6 hours after onset of symptoms and nearly 100% 10-12 hours after the event. High voltage electrophoresis has been used for rapid identification of patients with AMI; however, serum 157
Plasma Oxidase in Myocardial Infarction
enzyme cleavage of thermal lysine amino acids from CPK-MM and CPK-MB released by infarcted myocytes occurs after several hours. Therefore, most laboratories still use CPK-MB as the marker for AMI. As indicated previously, oxidase is released from the PMNs at the onset of ischemia or AMI, and its increased plasma activity can be detected immediately after the event. 3 With advances in thrombolytic therapy and percutaneous transluminal coronary angioplasty, the measurement of POA at the onset of symptoms may ensure that appropriate therapeutic measures are undertaken. In addition, POA may help the clinician differentiate between angina and AMI. In this report, we present a method of quantitating the POA in patients with AMI. The objectives of the work are threefold: (1) to determine the sensitivity of the method by assessing the magnitude of POA and comparing it to that observed in patients with AMI and angina pectoris as well as in healthy volunteers; (2) to assess the specificity rate of POA in patients with AMI and compare it to that in patients with acute infection; and (3) to establish that POA is detected much earlier than CPK-MB and is diagnostic for AMI then. Methods Patients. A total of 58 patients (42 men and 16
women, mean age 58 years; range 41-77 years) were enrolled in the study. An additional 12 healthy volunteers (6 men and 6 women, mean age 43 years) served as control subjects. The study population was divided into two groups. Group 1 consisted of 50 patients with possible AMI who were admitted to the coronary care unit for monitoring, including serial CPK-MB and POA. This group allowed us to assess the sensitivity rate of each assay for early diagnosis of AMI. Group 2 included 8 patients with pneumonia whose leukocyte counts were greater than 15,000 .uL. The results were used to determine the specificity rate of the POA assay. All patients who were admitted to the coronary care unit had initial and serial electrocardiograms for a minimum of 3 days. Chest roentgenograms were taken on admission and subsequently as required. Blood samples were obtained immediately after the decision was made to admit the patient to the coronary care unit, and those were used for routine laboratory assays, CPK and lactate dehydrogenase isoenzymes, andforPOA. Serum activities of CPK and CPK-MB isoenzyme were determined on admission and at 12-hour intervals for 36 hours. Measurement of those activities was accomplished by Helena Ret (electrophoresis) and Beckman methods, respectively. The normal range was 100269 IU/L for total CPK and 0-5% for the MB fraction. To assess the time-dependent changes in POA, daily determinations of enzymatic activity were performed for 5 days in 7 patients with MI. 158
Plasma Oxidase Activity Assay. The blood specimens (10 mL) for determination of POA were centrifuged at 3,000 revolutions per minute for 20 minutes at 4 0 C. Plasma was separated and filtered immediately through a 0.22-.u hydrophilic filter. The samples then either were processed immediately or stored at 4 0 C for late processing. The plasma (3 mL) was diluted with deionized water (1:1) and divided in 2 3-mL aliquots. Fifty microliters of 100 roM epinephrine hydrochloride (Sigma, St. Louis, MO) were added to one of the plasma samples and 50 .uL of deionized water to the other sample (control), and the cuvettes were incubated in a water bath at 37 0 C for 2 hours. After a 15-minute cooling period at room temperature (20 0 C), absorbance was measured spectrophotometrically at 480 nM (Respon~e Series, Gilford UV-VIS Spectrophotometer, Ciba-Coming, Oberlin, OH). The difference in absorbance between the sample incubated with epinephrine and that of the control was calculated. The adrenochrome concentration, as a measure of POA, was determined from a standard curve. Under these conditions, 1 unit of oxidase activity (U/L) represents l.uM of adrenochrome. Validation of Sensitivity and Specificity Rates of the Assay. The sensitivity rate of the assay was evaluated
by measuring POA and comparing it with standard CPK and CPK-MB determinations on serial samples as a proof of MI. The POA of these patients was compared with that of healthy volunteers. The specificity rate of the method was evaluated by comparing patients with myocardial ischemia and infarction to patients with other inflammatory processes in which neutrophil activation was believed to be present. Patients on immunosuppressive therapy and those with immunosuppressive states were excluded from the study. Statistical Analysis. Data are presented in the text, tables, and figures as mean ± standard error of the mean. Statistical evaluation was performed by Student's t-test for independence. A probability value of P less than 0.05 was considered statistically significant. Results
A summary of the subjects enrolled in the study, including demographic characteristics and CPK and POA values, is shown in Table L The POA was 84 ± 5 units for the control group, 127 ± 5 units for patients with angina and no AMI, 233 ± 13 units for patients with AMI, and 214 ± 10 units for patients with infections. On admission, patients with AMI had a threefold higher POA level (Figure 1) than that in healthy volunteers (P < 0.0001). The POA level in the group with angina pectoris was significantly lower than that in patients with AMI (P < 0.0001) (Figure 1), a finding that enhances the diagnostic capability of the assay to differentiate angina from AMI very early after the onset of chest pain. Plasma oxidase activity values in patients with infections (leukocytosis of> 15,000 .uL) also were September 1994 Volume 308 Number 3
Lasala et al
Table 1. Summary of Demographic Characteristics for the Four Groups
No. of Subjects MI Angina Controls Infections
22 28 12 8
Age 59.6 ± 58.8 ± 42.8 ± 56.3 ±
1.6 2 5 5
Male
Female
16 20 6 6
6 8 6 2
Admission CPK (lUlL)
Admission (%MB)
12-hour CPK (lUlL)
12-hour %MB
475 ± 81 298 ± 80
4.8 ± 1.6 0.3 ± 0.3
923 ± 13 311 ± 81
13 ± 1.3 0.3 ± 0.3
Admission ' POA (UlL) 233 ± 127 ± 84± 214 ±
13 5 5 10
CPK = creatine phosphokinase; MB = isoenzyme; MI = myocardial infarction; POA = plasma oxidase activity. Serum CPK and CPK-MB determinations were made on admission and 12 hours later in patients with acute MI and angina. The POA levels in patients with angina and acute MI were determined on admission, and those found to be greater than 200 UIL were diognostic for acute MI (See Results Section). Values are expressed in mean ± standard error of the mean.
elevated compared with those in control subjects and angina patients, but the level was somewhat lower than those observed in patients with AMI (not significant) (Figure 1)_ With one exception, for each patient, the initial specimen with a POA level greater than 200 U /L was considered diagnostic for AMI. The value of POA in the lone exception was compatible with those found in the groups with angina. The range of POA in the AMI group (excluding the above exception) was from 200422 U /L_ One patient had POA activity of 422 U /L, two others 341 and 309 U /L, and the remainder from 200-300 U fL. The high values in these three patients might have been due to a larger infarct, but this assumption is purely speculative because we do not know yet whether POA activity can be correlated with the size of the infarcted cardiac tissue. These findings merit further investigations. The range of the POA in the angina group (with one exception-197 U/L) was from 100-155 U/L. This patient had unstable angina and might have had a small infarct, because subsequently, there was an elevation of CPK-MB levels, but was not diagnostic for AMI. The diagnostic sensitivity rate of the POA assay as determined from the first blood sample obtained on admission to the coronary care unit being positive for
300
iii Plasma
AMI was 95% (ie, of the 22 patients, 21 had positive POA values for AMI). The CPK-MB from the same specimens was diagnostic in only 12 of 22 patients, thereby having a sensitivity rate of 54.5%. The mean time from the onset of chest pain to blood sampling in this group was 3.1 hours; this interval was known in 14 patients (64%). As indicated previously (also see Figure 1), patients with infections had elevated POA levels (range, 172266 U/L); therefore, the assay specificity rate in such conditions has no diagnostic value for angina or AMI. Follow-up of POA levels for 5 days in 7 patients with AMI indicates that those levels remain elevated longer than do those normally observed for serial CPK-MB; in some cases, this finding may help the physician make a retrospective diagnosis (Figure 2). Discussion
Although precise methods of calculating the extent of myocardial injury have not been derived, previous studies established a sound theoretical basis for the sensitivity and specificity rates of serial CPK-MB determinations for AMI. Deficiency ofthis assay is con-
Control vs MI: p < 0.0001 Angina vs MI: p < 0.0001 Control vs angina: p < 0.01
Oxidase Activity
::::;- 250
2>.
200
~
li 150
..
<
~,
100
"C
X
0
50 0 Controls
,Angina
, MI
" Intap.tions
Figure 1. Mean (± standard error of the me~n} plasma oxidase activity (POA) expressed in U/L in the four groups studied. Ml:,;, myocardial infarction. THE AMERICAN JOURNAL OF .THE MEDICAL SCIENCES
Figure 2. Time-dependent changes in plasma activity in seven patients after myocardial infarction (MI). Plasma oxidase activity (POA) is expressed in U /L~ Note that plasma oxidase activity stayed elevated longer after acute myocardial infarction than normally observed for serial creatine phosphokinase-MB (see the Results and Discussion sections)..
159
Plasma Oxidase in Myocardial Infarction
fined to the delayed release of the cardiac protein (CPK-MB) after AMI. Using data accumulated in this study, it is seen that POA after an ischemic episode or an infarction of cardiac muscle can be detected very shortly after the event. On the basis of our general knowledge at the onset of the ischemic episode, neutrophils become activated and obstruct capillary vessels, thereby causing microcirculatory changes and release injurious mediators before cytolysis of myocytes.6-10 As a period of ischemia is maintained, however, there appears the first clear evidence in our experience of a dissociation between diagnostic plasma increase of POA and that ofCPK-MB. 1O- 12 Later, when necrosis takes place, PMN infiltration augments further myocardial damage by releasing proteolytic enzymes and oxyradicals during phagocytosisY Therefore, it would be logical to expect that POA levels would be elevated as long as PMN activity is present in the infarcted myocardium. This was the case in examining the POA activity of 7 patients for 5 days. In contrast, diagnostic CPK-MB elevation persists no longer than 2-2112 days.14 During the evolution of MI, jeopardized zones not yet irreversibly damaged coexist with irreversibly damaged zones. Polymorphonuclear leukocyte activation with later infiltration occurs in both jeopardized and damaged areas. Attempts to evaluate PMN infiltration were made in animal models with other substances, such as myeloperoxidase, and a preliminary report indicated that adrenochrome, as a measure of oxidase activity, also may help detect MI. 3- 6 With our results, an increase in oxidase activity was found not only in patients with MI but also in patients with angina who did not have AMI, suggesting neutrophil activation with oxidase release. Plasma oxidase activity level was significantly higher in AMI than in angina, indicating a higher degree of enzymatic activity in AMI. This difference in oxidase activity probably reflects the various degrees of neutrophil activation and different severity of cellular injury reflecting the dynamic nature of the process. We assume that variations of the infarct size account for higher variability of POA within this group, as reflected by a higher standard error in the MI group compared with that in the angina and control groups. Despite this variability, the POA exceeding 200 UI L was 95% diagnostic for AMI. Currently, the magnitude of POA cannot be correlated with the infarct size. Further studies might provide such an answer. Use of serum POA changes as a marker for angina or AMI assumes that one compartment model applies to the system. In other words, the plasma space is considered the compartment of interest. The amount of adrenaline released might be viewed as an activator of the adrenochrome pathway executed by the stimulated PMN. It is implied that there could be increased adrenaline metabolism in patients with angina, AMI, ,and infections. This could translate into an increased 160
POA in response to higher endogenously released adrenaline. In resting humans, the concentration of adrenaline in the peripheral venous blood is on the order of 0.1-10 nM, and in AMI, it can attain values of 100 nM.lS-17 Because the adrenaline released from the adrenal gland reaches the coronary circulation without significant catabolism by the lungs, a portion of it therefore might be metabolized to adrenochrome by the PMN infiltration into the infarcted myocardium. In practical terms, this might represent a natural defense mechanism against arrhythmogenic properties of adrenaline, especially in the course of AMI. The increase in POA in patients with pneumonia reflects neutrophil activation by the inflammatory process. Therefore, the specificity rate of the test is decreased by this process, limiting the value of the test in patients with acute infections and other inflammatory processes. Serum CPK activity exceeds the normal range within 4-8 hours after the onset of AMI. 1O- 12 The peak CPK activity varies considerably, ranging from 8-58 hours later (mean, approximately 24 hours).14 Normal CPK was noted in 10 of the patients at the time of admission who later were proven to have AMI, with a resultant sensitivity rate of 54%. At the same time, POA was elevated significantly in '21 patients who later were proven to have MI as compared with the control group (sensitivity rate of 95%). Although POA was elevated to a lesser degree in one patient who had AMI, the value was in the upper range for those found in the angina group. The concept of a neutrophil origin of POA is supported by Matthews et al, 2 who demonstrated release of oxidase by stimulating neutrophils in vitro. Other experimental studies in animals, however, failed to demonstrate neutrophil infiltration or significant myeloperoxidase activity as a measure of neutrophil activation after 48 hours of MJ.7 In patients in whom samples were obtained for 5 days, 1 of the 7 underwent cardiac catheterization and angioplasty on the third day after MI. The POA level was noted to increase further after the procedure, which shifted the POA curve upward (Figure 2). The increase in POA level supports other studies of increased PMN activation after reperfusion.6-9 Two patients received thrombolytics in the emergency room before samples were obtained for the study, and although they were not included in the study, samples for POA assay were obtained 4 hours after administration of the thrombolytics. The oxidase activity in these samples was approximately three times that of the mean for AMI patients. The increase in POA level observed in these three patients who had reperfusion supports the hypothesis that an oxidative mechanism might be responsible for reperfusion injury.IS Although the study was not designed to investigate reperfusion injury, the information obtained is of importance for the design of new investigations. September 1994 Volume 308 Number 3
Lasala et al
Early identification of patients with angina pectoris and AMI is a primary goal for the clinician encountering a patient with chest pain and nondiagnostic electrocardiogram. Because the sensitivity rate of POA assay for AMI was 95% on admission and also could identify those patients with angina, this method could represent an alternative and unique way to screen for AMI or angina in patients who come to emergency rooms with chest pain but with no evidence of acute infections. Under the circumstances that this study was conducted, the sensitivity rate of POA assay was significantly greater than that of CPK-MB. Application of POA assay can permit rapid identification of the patient with angina and AMI, thereby improving use of scarce coronary care unit resources and decreasing financial burden. In addition, identifying patients with AMI with nondiagnostic electrocardiogram before precipitate release from the emergency room would afford them the opportunity to benefit from thrombolytic therapy or percutaneous transluminal coronary angioplasty. Finally, the findings in this study do not imply that the assay should be used to make a clinically definitive diagnosis for AMI, because the assay is not specific and only measures the degree of PMN activation as expressed by the POA during angina episodes and AMI. However, even with the limited number of patients enrolled in this study, it was possible to differentiate between angina pectoris and AMI as well as to diagnose AMI (in patients without acute infections) before the time that CPK-MB values became diagnostic. References 1. Fentone JC, Ward PA. Polymorphonuclear leukocytes-Mediated cell and tissue imaging: Oxygen metabolites and their relation to human disease. Hum Pathol. 1985;16:973-8. 2. Matthews SB, Henderson AH, Campbell AK. The adrenochrome pathway: The major route for adrenaline catabolism by polymorphonuclear leukocytes. J Mol Cell Cardio!. 1985;17: 339-48. 3. Matthews SB, Campbell AK. Neutrophil activation after myocardial infarction (letter). Lancet. 1984;2:756-7.
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
4. Matthews SB, Hallett ME, Henderson AH, Campbell AK. The adrenochrome pathway: A potential catabolic route for adrenaline metabolism in inBammatory disease. Advances in Myocardiology. 1984;6:367-81. 5. Matthews SB, Henderson AH, Campbell AK. The oxidation of adrenaline to adrenochrome by polymorphonuclear leucocytes. Biochem Soc Trans. 1983;II:191. 6. Hoshida S, Kuzuya T, Nishida M, Kim Y, Kitabatake A, Kamada T, et aI. Attenuation of neutrophil function by in· hibitors of arachidonate metabolism reduces the extent of canine myocardial infarction. Am J Cardio!. 1989;63:24E-8E. 7. Smith EF m, Egan JW, Bugelski PJ, Hillegass LM, Hill DE, Griswold DE. Temporal relation between neutrophil accumulation and myocardial reperfusion injury. Am J Physio!. 1988;255:H1060-8. 8. Romson JL, Jolly SR, Lucchesi BR. Protection of ischemic myocardium by pharmacologic manipulation of leukocyte function. Cardiovascular Reviews and Reports. 1992;313:21-35. 9. Goetzl EJ, Pickett WC. The human PMN leukocyte chemo· tactic activity of complex hydroxy-eicosatetraenoic acids (HETEs). J Immuno!. 1980;125:1789-91. 10. Delaghe J, De Buyxere M, De Sheerder I, Vandebogaerde J, Van den Abeele AM, Gheeraert P. Creatine determinations as an early marker for the diagnosis of acute myocardial infarction. Ann Clin Biochem. 1988;25:383-8. " " 11. Collison PO, Rosalki SB, Flather M, Wolman R, Evans T. Early diagnosis of myocardial infarc40n by timed sequential enzyme measurements. Ann Clin Biochem. 1988;25:376-82. 12. Dufour RD, LaGrenade A, Guerra J. Rapid serial" enzyme measurements in evaluation of patients with suspectedmyocardial infarction. Am J Cardio!. 1989;63:652-5. 13. Bellavite P. The superoxide-forming enzymatic system of phagocytes. Free Radic BioI Med. 1988;4:225-61. 14. Lee TH, Goldman L. Serum enzyme assays in the diagnosis of acute myocardial infarction. Ann Intern Med. 1986;105:221. 15. De Champlain J, Farley L, Cousineau D, Van Ameringen M. Circulating catecholamine levels in human and experimental hypertension. Circ Res. 1976;382:109-14. 16. Sapira JD, Klaniecki T, Rizk M. Modified Buorometric method for determining plasma catecholamines. Clin Chem. 1971;17:486-91. 17. Benedict CR, Grahame Smith DG. Plasma adrenaline and noradrenaline concentrations and dopamine B hydroxylase activity in myocardial infarction with and without cardiogenic shock. Br Heart J~ 1979;42:214-20. 18. Litt MR, Jeremy RW, Weisman HF, Winkelstein JA, Becker LC. Neutrophil depletion limited to reperfusion reduces myocardial infarct size after 90 minutes of ischemia. Evidence for neutrophil mediated reperfusion injury. Circulation. 1989;80: 1816-27.
161
ABSTRACT: The availability of techniques such as surgical reperfusion, angioplasty, and thrombolysis for the treatment of acute myocardial infarction (AMI) has revived interest in seeking an early detectable biochemical marker diagnostic for AMI. Therefore, we investigated whether an unidentified oxidase that is released by activated neutrophils at the onset of AMI could be used as an early diagnostic assay. The conversion by plasma oxidase of 1 ~M of adrenaline to 1 ~M of adrenochrome represents the plasma oxidase activity (POA) of 1 UIL. Fifty patients suspected of having AMI, 40% of whose electrocardiograms were nondiagnostic for AMI, were admitted to the coronary care unit, and venous blood samples were obtained for determination of the POA and creatine phosphokinaseMB levels. Healthy volunteers (n = 12) served as control subjects, and 8 patients with pneumonia whose leukocyte counts were greater than 15,000 ~L were included in the study. In those with AMI (n = 22), as determined by serial creatine phosphokinase-MB, the mean POA (± standard error of the mean) was 233 ± 13 UIL, and in those with angina and no AMI (n = 28) was 127 ± 5 UIL (P < 0.0001). In the control group, mean POA (± standard error of the mean) was 84 ± 5 UIL (control versus angina; P < 0.01) and for those with infection was 214 ± 10 UIL. At admission, the creatine phosphokinase-MB was diagnostic for only 12 of the 22 patients with AMI (sensitivity rate of 54%), whereas in 21 of those patients, the POA values were diagnostic for AMI (sensitivity rate of95%). Determination of POA may represent an alternative sensitive From the University of Mississippi Medical Center, Jackson, Mississippi. Presented at the 45th Annual Meeting of the Southern Society for ClinicalInvestigation, New Orleans, Louisiana, January 30-February 1, 1991 (Clin Res 1991; 39[4]:799A). Correspondence: Angel K. Markov, MD, Department of Medicine, Division of Cardiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
method to screen for AMI or ischemia in patients with anginal symptoms and no evidence of acute infection. KEY INDEXING TERMS: Myocardial infarction; Oxidase; Assay; Screening. [Am J Med Sci 1994;308(3):157-161.]
P
olymorphonuclear leukocyte (PMN)-deriyed oxyradicals have been shown to contribute to the tissue injury associated with acute myocardial infarction (AMI).l In addition, activated PMNs release other potent substances that likely have deleterious effects (proteases, leukotrienes, thromboxane, and others). In inflammatory conditions in which PMN tissue infiltration occurs, there is evidence that the activated PMN can oxidize adrenaline to adrenochrome. 2 - s Medium isolated after stimulation of the PMN retained its ability to oxidize adrenaline to adrenochrome. 2 - 4 Plasma from patients with inflammatory diseases causes more oxidation of adrenaline to adrenochrome than do the sera from healthy volunteers. 2 - 4 Matthews et al2-4 demonstrated the presence of an adrenaline oxidase in patients with acute inflammatory conditions as a biochemical marker for plasma oxidase activity (POA). Although the role of the oxidase is not defined, it may act as a natural defense mechanism against endogenously released adrenaline in the course of myocardial infarction (MI), thereby diminishing the likelihood for occurrence of ventricular arrhythmias. Creatinine phosphokinase (CPK) consists of 2 subunits, M and B, which form 3 isoenzymes denoted as CPK-MM, CPK-BB, and CPK-MB. The latter isoenzyme usually is found in the cardiac muscle, but it could be detected in small quantities in other tissue. Although CPK-MB has no absolute specificity rate, an elevated level in serum generally is accepted as the best indicator for AMI. The sensitivity and specificity rates by serial CPK-MB sampling approach 90% 5-6 hours after onset of symptoms and nearly 100% 10-12 hours after the event. High voltage electrophoresis has been used for rapid identification of patients with AMI; however, serum 157
Plasma Oxidase in Myocardial Infarction
enzyme cleavage of thermal lysine amino acids from CPK-MM and CPK-MB released by infarcted myocytes occurs after several hours. Therefore, most laboratories still use CPK-MB as the marker for AMI. As indicated previously, oxidase is released from the PMNs at the onset of ischemia or AMI, and its increased plasma activity can be detected immediately after the event. 3 With advances in thrombolytic therapy and percutaneous transluminal coronary angioplasty, the measurement of POA at the onset of symptoms may ensure that appropriate therapeutic measures are undertaken. In addition, POA may help the clinician differentiate between angina and AMI. In this report, we present a method of quantitating the POA in patients with AMI. The objectives of the work are threefold: (1) to determine the sensitivity of the method by assessing the magnitude of POA and comparing it to that observed in patients with AMI and angina pectoris as well as in healthy volunteers; (2) to assess the specificity rate of POA in patients with AMI and compare it to that in patients with acute infection; and (3) to establish that POA is detected much earlier than CPK-MB and is diagnostic for AMI then. Methods Patients. A total of 58 patients (42 men and 16
women, mean age 58 years; range 41-77 years) were enrolled in the study. An additional 12 healthy volunteers (6 men and 6 women, mean age 43 years) served as control subjects. The study population was divided into two groups. Group 1 consisted of 50 patients with possible AMI who were admitted to the coronary care unit for monitoring, including serial CPK-MB and POA. This group allowed us to assess the sensitivity rate of each assay for early diagnosis of AMI. Group 2 included 8 patients with pneumonia whose leukocyte counts were greater than 15,000 .uL. The results were used to determine the specificity rate of the POA assay. All patients who were admitted to the coronary care unit had initial and serial electrocardiograms for a minimum of 3 days. Chest roentgenograms were taken on admission and subsequently as required. Blood samples were obtained immediately after the decision was made to admit the patient to the coronary care unit, and those were used for routine laboratory assays, CPK and lactate dehydrogenase isoenzymes, andforPOA. Serum activities of CPK and CPK-MB isoenzyme were determined on admission and at 12-hour intervals for 36 hours. Measurement of those activities was accomplished by Helena Ret (electrophoresis) and Beckman methods, respectively. The normal range was 100269 IU/L for total CPK and 0-5% for the MB fraction. To assess the time-dependent changes in POA, daily determinations of enzymatic activity were performed for 5 days in 7 patients with MI. 158
Plasma Oxidase Activity Assay. The blood specimens (10 mL) for determination of POA were centrifuged at 3,000 revolutions per minute for 20 minutes at 4 0 C. Plasma was separated and filtered immediately through a 0.22-.u hydrophilic filter. The samples then either were processed immediately or stored at 4 0 C for late processing. The plasma (3 mL) was diluted with deionized water (1:1) and divided in 2 3-mL aliquots. Fifty microliters of 100 roM epinephrine hydrochloride (Sigma, St. Louis, MO) were added to one of the plasma samples and 50 .uL of deionized water to the other sample (control), and the cuvettes were incubated in a water bath at 37 0 C for 2 hours. After a 15-minute cooling period at room temperature (20 0 C), absorbance was measured spectrophotometrically at 480 nM (Respon~e Series, Gilford UV-VIS Spectrophotometer, Ciba-Coming, Oberlin, OH). The difference in absorbance between the sample incubated with epinephrine and that of the control was calculated. The adrenochrome concentration, as a measure of POA, was determined from a standard curve. Under these conditions, 1 unit of oxidase activity (U/L) represents l.uM of adrenochrome. Validation of Sensitivity and Specificity Rates of the Assay. The sensitivity rate of the assay was evaluated
by measuring POA and comparing it with standard CPK and CPK-MB determinations on serial samples as a proof of MI. The POA of these patients was compared with that of healthy volunteers. The specificity rate of the method was evaluated by comparing patients with myocardial ischemia and infarction to patients with other inflammatory processes in which neutrophil activation was believed to be present. Patients on immunosuppressive therapy and those with immunosuppressive states were excluded from the study. Statistical Analysis. Data are presented in the text, tables, and figures as mean ± standard error of the mean. Statistical evaluation was performed by Student's t-test for independence. A probability value of P less than 0.05 was considered statistically significant. Results
A summary of the subjects enrolled in the study, including demographic characteristics and CPK and POA values, is shown in Table L The POA was 84 ± 5 units for the control group, 127 ± 5 units for patients with angina and no AMI, 233 ± 13 units for patients with AMI, and 214 ± 10 units for patients with infections. On admission, patients with AMI had a threefold higher POA level (Figure 1) than that in healthy volunteers (P < 0.0001). The POA level in the group with angina pectoris was significantly lower than that in patients with AMI (P < 0.0001) (Figure 1), a finding that enhances the diagnostic capability of the assay to differentiate angina from AMI very early after the onset of chest pain. Plasma oxidase activity values in patients with infections (leukocytosis of> 15,000 .uL) also were September 1994 Volume 308 Number 3
Lasala et al
Table 1. Summary of Demographic Characteristics for the Four Groups
No. of Subjects MI Angina Controls Infections
22 28 12 8
Age 59.6 ± 58.8 ± 42.8 ± 56.3 ±
1.6 2 5 5
Male
Female
16 20 6 6
6 8 6 2
Admission CPK (lUlL)
Admission (%MB)
12-hour CPK (lUlL)
12-hour %MB
475 ± 81 298 ± 80
4.8 ± 1.6 0.3 ± 0.3
923 ± 13 311 ± 81
13 ± 1.3 0.3 ± 0.3
Admission ' POA (UlL) 233 ± 127 ± 84± 214 ±
13 5 5 10
CPK = creatine phosphokinase; MB = isoenzyme; MI = myocardial infarction; POA = plasma oxidase activity. Serum CPK and CPK-MB determinations were made on admission and 12 hours later in patients with acute MI and angina. The POA levels in patients with angina and acute MI were determined on admission, and those found to be greater than 200 UIL were diognostic for acute MI (See Results Section). Values are expressed in mean ± standard error of the mean.
elevated compared with those in control subjects and angina patients, but the level was somewhat lower than those observed in patients with AMI (not significant) (Figure 1)_ With one exception, for each patient, the initial specimen with a POA level greater than 200 U /L was considered diagnostic for AMI. The value of POA in the lone exception was compatible with those found in the groups with angina. The range of POA in the AMI group (excluding the above exception) was from 200422 U /L_ One patient had POA activity of 422 U /L, two others 341 and 309 U /L, and the remainder from 200-300 U fL. The high values in these three patients might have been due to a larger infarct, but this assumption is purely speculative because we do not know yet whether POA activity can be correlated with the size of the infarcted cardiac tissue. These findings merit further investigations. The range of the POA in the angina group (with one exception-197 U/L) was from 100-155 U/L. This patient had unstable angina and might have had a small infarct, because subsequently, there was an elevation of CPK-MB levels, but was not diagnostic for AMI. The diagnostic sensitivity rate of the POA assay as determined from the first blood sample obtained on admission to the coronary care unit being positive for
300
iii Plasma
AMI was 95% (ie, of the 22 patients, 21 had positive POA values for AMI). The CPK-MB from the same specimens was diagnostic in only 12 of 22 patients, thereby having a sensitivity rate of 54.5%. The mean time from the onset of chest pain to blood sampling in this group was 3.1 hours; this interval was known in 14 patients (64%). As indicated previously (also see Figure 1), patients with infections had elevated POA levels (range, 172266 U/L); therefore, the assay specificity rate in such conditions has no diagnostic value for angina or AMI. Follow-up of POA levels for 5 days in 7 patients with AMI indicates that those levels remain elevated longer than do those normally observed for serial CPK-MB; in some cases, this finding may help the physician make a retrospective diagnosis (Figure 2). Discussion
Although precise methods of calculating the extent of myocardial injury have not been derived, previous studies established a sound theoretical basis for the sensitivity and specificity rates of serial CPK-MB determinations for AMI. Deficiency ofthis assay is con-
Control vs MI: p < 0.0001 Angina vs MI: p < 0.0001 Control vs angina: p < 0.01
Oxidase Activity
::::;- 250
2>.
200
~
li 150
..
<
~,
100
"C
X
0
50 0 Controls
,Angina
, MI
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Figure 1. Mean (± standard error of the me~n} plasma oxidase activity (POA) expressed in U/L in the four groups studied. Ml:,;, myocardial infarction. THE AMERICAN JOURNAL OF .THE MEDICAL SCIENCES
Figure 2. Time-dependent changes in plasma activity in seven patients after myocardial infarction (MI). Plasma oxidase activity (POA) is expressed in U /L~ Note that plasma oxidase activity stayed elevated longer after acute myocardial infarction than normally observed for serial creatine phosphokinase-MB (see the Results and Discussion sections)..
159
Plasma Oxidase in Myocardial Infarction
fined to the delayed release of the cardiac protein (CPK-MB) after AMI. Using data accumulated in this study, it is seen that POA after an ischemic episode or an infarction of cardiac muscle can be detected very shortly after the event. On the basis of our general knowledge at the onset of the ischemic episode, neutrophils become activated and obstruct capillary vessels, thereby causing microcirculatory changes and release injurious mediators before cytolysis of myocytes.6-10 As a period of ischemia is maintained, however, there appears the first clear evidence in our experience of a dissociation between diagnostic plasma increase of POA and that ofCPK-MB. 1O- 12 Later, when necrosis takes place, PMN infiltration augments further myocardial damage by releasing proteolytic enzymes and oxyradicals during phagocytosisY Therefore, it would be logical to expect that POA levels would be elevated as long as PMN activity is present in the infarcted myocardium. This was the case in examining the POA activity of 7 patients for 5 days. In contrast, diagnostic CPK-MB elevation persists no longer than 2-2112 days.14 During the evolution of MI, jeopardized zones not yet irreversibly damaged coexist with irreversibly damaged zones. Polymorphonuclear leukocyte activation with later infiltration occurs in both jeopardized and damaged areas. Attempts to evaluate PMN infiltration were made in animal models with other substances, such as myeloperoxidase, and a preliminary report indicated that adrenochrome, as a measure of oxidase activity, also may help detect MI. 3- 6 With our results, an increase in oxidase activity was found not only in patients with MI but also in patients with angina who did not have AMI, suggesting neutrophil activation with oxidase release. Plasma oxidase activity level was significantly higher in AMI than in angina, indicating a higher degree of enzymatic activity in AMI. This difference in oxidase activity probably reflects the various degrees of neutrophil activation and different severity of cellular injury reflecting the dynamic nature of the process. We assume that variations of the infarct size account for higher variability of POA within this group, as reflected by a higher standard error in the MI group compared with that in the angina and control groups. Despite this variability, the POA exceeding 200 UI L was 95% diagnostic for AMI. Currently, the magnitude of POA cannot be correlated with the infarct size. Further studies might provide such an answer. Use of serum POA changes as a marker for angina or AMI assumes that one compartment model applies to the system. In other words, the plasma space is considered the compartment of interest. The amount of adrenaline released might be viewed as an activator of the adrenochrome pathway executed by the stimulated PMN. It is implied that there could be increased adrenaline metabolism in patients with angina, AMI, ,and infections. This could translate into an increased 160
POA in response to higher endogenously released adrenaline. In resting humans, the concentration of adrenaline in the peripheral venous blood is on the order of 0.1-10 nM, and in AMI, it can attain values of 100 nM.lS-17 Because the adrenaline released from the adrenal gland reaches the coronary circulation without significant catabolism by the lungs, a portion of it therefore might be metabolized to adrenochrome by the PMN infiltration into the infarcted myocardium. In practical terms, this might represent a natural defense mechanism against arrhythmogenic properties of adrenaline, especially in the course of AMI. The increase in POA in patients with pneumonia reflects neutrophil activation by the inflammatory process. Therefore, the specificity rate of the test is decreased by this process, limiting the value of the test in patients with acute infections and other inflammatory processes. Serum CPK activity exceeds the normal range within 4-8 hours after the onset of AMI. 1O- 12 The peak CPK activity varies considerably, ranging from 8-58 hours later (mean, approximately 24 hours).14 Normal CPK was noted in 10 of the patients at the time of admission who later were proven to have AMI, with a resultant sensitivity rate of 54%. At the same time, POA was elevated significantly in '21 patients who later were proven to have MI as compared with the control group (sensitivity rate of 95%). Although POA was elevated to a lesser degree in one patient who had AMI, the value was in the upper range for those found in the angina group. The concept of a neutrophil origin of POA is supported by Matthews et al, 2 who demonstrated release of oxidase by stimulating neutrophils in vitro. Other experimental studies in animals, however, failed to demonstrate neutrophil infiltration or significant myeloperoxidase activity as a measure of neutrophil activation after 48 hours of MJ.7 In patients in whom samples were obtained for 5 days, 1 of the 7 underwent cardiac catheterization and angioplasty on the third day after MI. The POA level was noted to increase further after the procedure, which shifted the POA curve upward (Figure 2). The increase in POA level supports other studies of increased PMN activation after reperfusion.6-9 Two patients received thrombolytics in the emergency room before samples were obtained for the study, and although they were not included in the study, samples for POA assay were obtained 4 hours after administration of the thrombolytics. The oxidase activity in these samples was approximately three times that of the mean for AMI patients. The increase in POA level observed in these three patients who had reperfusion supports the hypothesis that an oxidative mechanism might be responsible for reperfusion injury.IS Although the study was not designed to investigate reperfusion injury, the information obtained is of importance for the design of new investigations. September 1994 Volume 308 Number 3
Lasala et al
Early identification of patients with angina pectoris and AMI is a primary goal for the clinician encountering a patient with chest pain and nondiagnostic electrocardiogram. Because the sensitivity rate of POA assay for AMI was 95% on admission and also could identify those patients with angina, this method could represent an alternative and unique way to screen for AMI or angina in patients who come to emergency rooms with chest pain but with no evidence of acute infections. Under the circumstances that this study was conducted, the sensitivity rate of POA assay was significantly greater than that of CPK-MB. Application of POA assay can permit rapid identification of the patient with angina and AMI, thereby improving use of scarce coronary care unit resources and decreasing financial burden. In addition, identifying patients with AMI with nondiagnostic electrocardiogram before precipitate release from the emergency room would afford them the opportunity to benefit from thrombolytic therapy or percutaneous transluminal coronary angioplasty. Finally, the findings in this study do not imply that the assay should be used to make a clinically definitive diagnosis for AMI, because the assay is not specific and only measures the degree of PMN activation as expressed by the POA during angina episodes and AMI. However, even with the limited number of patients enrolled in this study, it was possible to differentiate between angina pectoris and AMI as well as to diagnose AMI (in patients without acute infections) before the time that CPK-MB values became diagnostic. References 1. Fentone JC, Ward PA. Polymorphonuclear leukocytes-Mediated cell and tissue imaging: Oxygen metabolites and their relation to human disease. Hum Pathol. 1985;16:973-8. 2. Matthews SB, Henderson AH, Campbell AK. The adrenochrome pathway: The major route for adrenaline catabolism by polymorphonuclear leukocytes. J Mol Cell Cardio!. 1985;17: 339-48. 3. Matthews SB, Campbell AK. Neutrophil activation after myocardial infarction (letter). Lancet. 1984;2:756-7.
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
4. Matthews SB, Hallett ME, Henderson AH, Campbell AK. The adrenochrome pathway: A potential catabolic route for adrenaline metabolism in inBammatory disease. Advances in Myocardiology. 1984;6:367-81. 5. Matthews SB, Henderson AH, Campbell AK. The oxidation of adrenaline to adrenochrome by polymorphonuclear leucocytes. Biochem Soc Trans. 1983;II:191. 6. Hoshida S, Kuzuya T, Nishida M, Kim Y, Kitabatake A, Kamada T, et aI. Attenuation of neutrophil function by in· hibitors of arachidonate metabolism reduces the extent of canine myocardial infarction. Am J Cardio!. 1989;63:24E-8E. 7. Smith EF m, Egan JW, Bugelski PJ, Hillegass LM, Hill DE, Griswold DE. Temporal relation between neutrophil accumulation and myocardial reperfusion injury. Am J Physio!. 1988;255:H1060-8. 8. Romson JL, Jolly SR, Lucchesi BR. Protection of ischemic myocardium by pharmacologic manipulation of leukocyte function. Cardiovascular Reviews and Reports. 1992;313:21-35. 9. Goetzl EJ, Pickett WC. The human PMN leukocyte chemo· tactic activity of complex hydroxy-eicosatetraenoic acids (HETEs). J Immuno!. 1980;125:1789-91. 10. Delaghe J, De Buyxere M, De Sheerder I, Vandebogaerde J, Van den Abeele AM, Gheeraert P. Creatine determinations as an early marker for the diagnosis of acute myocardial infarction. Ann Clin Biochem. 1988;25:383-8. " " 11. Collison PO, Rosalki SB, Flather M, Wolman R, Evans T. Early diagnosis of myocardial infarc40n by timed sequential enzyme measurements. Ann Clin Biochem. 1988;25:376-82. 12. Dufour RD, LaGrenade A, Guerra J. Rapid serial" enzyme measurements in evaluation of patients with suspectedmyocardial infarction. Am J Cardio!. 1989;63:652-5. 13. Bellavite P. The superoxide-forming enzymatic system of phagocytes. Free Radic BioI Med. 1988;4:225-61. 14. Lee TH, Goldman L. Serum enzyme assays in the diagnosis of acute myocardial infarction. Ann Intern Med. 1986;105:221. 15. De Champlain J, Farley L, Cousineau D, Van Ameringen M. Circulating catecholamine levels in human and experimental hypertension. Circ Res. 1976;382:109-14. 16. Sapira JD, Klaniecki T, Rizk M. Modified Buorometric method for determining plasma catecholamines. Clin Chem. 1971;17:486-91. 17. Benedict CR, Grahame Smith DG. Plasma adrenaline and noradrenaline concentrations and dopamine B hydroxylase activity in myocardial infarction with and without cardiogenic shock. Br Heart J~ 1979;42:214-20. 18. Litt MR, Jeremy RW, Weisman HF, Winkelstein JA, Becker LC. Neutrophil depletion limited to reperfusion reduces myocardial infarct size after 90 minutes of ischemia. Evidence for neutrophil mediated reperfusion injury. Circulation. 1989;80: 1816-27.
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