Response of serum indicators of myocardial infarction following exercise-induced muscle injury

Original Contributions

Response of Serum Indicators of Myocardial Infarction Following Exercise-Induced Muscle Injury REID HAYWARD, PHD,* JANICE M. BALOG, PHD,1CAROLE M. SCHNEIDER, PHD:I: The purpose of this investigation was to determine the response of three parameters used in the assessment of acute myocardial infarction (AMI) after a single bout of eccentric exercise designed to elicit skeletal muscle injury. Total creatine kinase (CK), CK-MB isoenzyme (CK-MB), and the leukocyte differential were determined after a 20-minute bench-stepping exercise in 21 men ranging in age from 30 to 45 years. Comparison of several criteria showed that the use of CK-MB or the relative lymphocyte percentage alone resulted in 11% and 1.8% respectively, of data collection points exceeding cutoff values suggestive of AMI. However, the use of both parameters in combination completely eliminated false-positive results with no data collection points meeting the criterion. It is thus suggested that CK-MB activity in conjunction with the relative lymphocyte percentage may not only provide incremental value in the detection of AMI but also reduce the incidence of misdiagnosis associated with exercise. (Am J Emerg Med 1998;16:107-113. Copyright © 1998 by W.B. Saunders Company) Skeletal muscle injury is a common phenomenon that may be actuated by a number of different factors including exercise, and has been noted as a complication in the assessment of individuals in whom acute myocardial infarction (AMI) is suspected. Skeletal muscle injury after exercise, particularly eccentric exercise, has been identified. 1,2 Indicators of skeletal muscle injury include increases in serum levels of muscle proteins? ,4 structural disruption of muscle fibers, 2,5 delayed-onset muscle soreness, 6,7 and changes in maximal voluntary muscular strength and contractile properties of muscle. 1,8 Of these indicators, increases in the serum activity of intramuscular enzymes may be of most concern to the clinician. This is because exercise-induced muscle injury mimics symptoms suggestive of AMI. Numerous investigators have shown that eccentric exercise results

From the *Human Performance Laboratory, University of Arkansas, and the -i-United States Department of Agriculture, Agricultural Research Service, Fayetteville, AR; and the :~Department of Kinesiology, University of Northern Colorado, Greeley, CO. Manuscript received September 19, 1996, returned October 21, 1996; accepted November 18, 1996. Address reprint requests to Dr Hayward, Department of Physiology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust St, Philadelphia, PA 19107-6799. Key Words: Eccentric contractions, creatine kinase, leukocytes, lymphocytes, myocardial infarction, muscle injury. Copyright © 1998 by W.B. Saunders Company 0735-6757/98/1602-0001 $8.00/0

in significant increases in serum levels of the creatine kinase-MB isoenzyme (CK-MB). 3,9,1° Such observations have led to the identification of diagnostic confusion in people who have recently been physically active and who have developed chest pain suggestive of myocardial ischemia.l~, 12 Although several new serum measures have been proposed, 13-15serum creatine kinase (CK) activity along with its isoenzymes and their subforms continue to be among the most widely implemented techniques in the diagnosis of AMI. Recently, however, assessment of the leukocyte differential count has seen a resurgence as an independent indicator of AMI. J6 Early methods used in the detection of AMI such as total CK and CK-MB activity have proven to be quite effective in assessing myocardial damage. However, these methods fail to differentiate between cardiac and skeletal muscle damage after exercise in apparently healthy individuals. This has led to the questioning of such measures to accurately assess myocardial damage in any individual who has recently been physically active. Recently developed methods, as would be expected, focus primarily on those individuals presenting symptoms suggestive of myocardial injury. Few studies have observed the response of these more recently used indicators of AMI after exercise-induced muscle injury. Therefore, the purpose of this investigation was to determine the response of three parameters used in the assessment of AMI, after a single bout of eccentric exercise designed to elicit skeletal muscle injury.

METHODS After approval from the University Institutional Review Board, 21 men ranging in age from 30 to 45 years volunteered as subjects. Before admission into the investigation, all participants were informed about the purpose, methods, anticipated benefits, and potential risks involved. Each subject read and signed an informed consent. Inclusion criteria were the absence of hypertension, diabetes, cardiovascular disease, alcohol dependence, and ongoing drug usage as determined by a health history questionnaire. Subjects were instructed to refrain from exercise during the 2 weeks before as well as the 96 hours after the exercise intervention. Subject age, height, and weight were 35 _+ 1.2 years, 187.9 + 2.3 cm, and 80.6 + 1.7 kg, respectively. All subjects reported to the Human Performance Laboratory on a specified morning at 0700 hours. Each subject completed a 107

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bench-stepping exercise similar to that used by other researchers, 1°,17-19 which was initiated at 0730. The exercise bout consisted of a bench-stepping test 20 minutes in duration, thus ending at approximately 0750 hours. The bench height was set at 50 cm. An electronic metronome sounded at 1-second intervals, at which time one step was completed. This resulted in a total of 15 cycles (60 steps) per minute and a total of 300 cycles (1,200 steps) during the 20-minute test. The exercise primarily allowed for eccentric contractions of the left limb knee extensors and right limb ankle plantar flexors. After the initial testing and exercise intervention, blood sampling was conducted at specified time intervals. These intervals included 0 (0800 hours), 6 (1400 hours), 12 (2000 hours), 24 (0800 hours), 48 (0800 hours), 72 (0800 hours), and 96 (0800 hours) hours postexercise. Blood samples were collected in two tubes, The first tube, containing EDTA as the anticoagulating agent, was stored at room temperature until leukocyte analysis was performed. In the second tube, blood was allowed to clot at room temperature and was centrifuged. Serum was removed and frozen at -20°C until analysis. Blood samples were assayed for activity of CK (Sigma Diagnostics Procedure UV-47), and CK-MB (Sigma Diagnostics Procedure UV-49) on the Express Plus automated clinical chemistry system (Ciba-Corning, Oberlin, OH). Total leukocyte and lymphocyte counts were determined using the CELL-DYN 3500SL automated hematology analyzer (Abbot Diagnostics, Abbot Park, IL). Normal values for CK and CK-MB activity are stated as 23-170 U/L and undetectable-10 U/L, respectively. Normal values for leukocytes and lymphocytes are stated as 4.8-10.8 × 109 cells/L and 0.96-3.40 × 109 cells/L, respectively. Statistical analysis. Data are reported as mean _+ SEM. Statistical analyses were conducted using a one-way analysis of variance (ANOVA) with repeated measures to determine whether significant differences occurred over time. After a significant F-ratio, differences from baseline values were identified by a Helmerk orthogonal contrast post hoc analysis. Pearson product moment correlations and linear regression analyses were conducted to determine the strength of relationship as well as variance accounted for between selected variables. All differences and correlations were considered significant at P < .05.

RESULTS Serum Enzyme Response CK activity increased significantly immediately after the exercise bout and remained significantly elevated at the 6, 12, and 24 hour intervals (Figure 1). Pre-exercise evaluation of total serum CK showed that two individuals (9.5%) began the investigation with activity values that exceeded the upper limit of normal (199 U/L and 212 U/L), which is not surprising because of the known individual variability seen in CK activity. Omission of these individuals lowered the range of pre-exercise values to between 64 U/L and 153 U/L, well within the normal range. Of the eight data collection intervals, seven presented mean values within the range of normal. The remaining interval (12 hours) resulted in a mean enzyme activity of 208 + 17.4 U/L, just above the upper limit of normal. During the data collection period, 62% of subjects (13 of 21) presented total CK activity above the normal range during at least one data collection interval. Of the 13 subjects who presented above normal CK activity, 85% (11 of 13) showed elevations at multiple data collection intervals that typically occurred at consecutive time periods. The remaining eight subjects did show increases in CK activity

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FIGURE 1. Serum CK response to the exercise bout. Values are mean _+ SEM. The solid box indicates the exercise intervention. *Significantly different from pre-exercise values. during the investigation, with an average increase of 53% from pre-exercise to peak values. However, the peak activity shown by these individuals did not exceed the normal range. Mean CK activity increased during the first 12 hours postexercise, with before 0, 0 to 6, and 6 to 12 hour increases of 17%, 24%, and 23%, respectively. Maximum mean CK activity occurred at 12 hours, resulting in an 80% increase from pre-exercise values. This increase was followed by a steady decline in total CK activity during the next 36 hours. CK activity decreased from the peak of 208.0 + 17.4 U/L to 146.7 + 12.8 U/L at 48 hours postexercise, resulting in a 30% decrease. Although a substantial decrease occurred at 48 hours, mean activity at this time period was still 27% higher than pre-exercise levels. During the final 48 hours of data collection, 57% (12 of 21) of subjects had a substantial increase in CK activity (69.1 U/L), whereas the remaining subjects maintained the existing downward trend. One hundred sixty-eight total CK assays were performed, of which 29% (49 of 168) resulted in values above the normal range. Before participation in the exercise intervention, CK-MB activity ranged from 6.0 U/L to 13.0 U/L, with two subjects showing values above the normal range (11 U/L, 13 U/L). These individuals were the subjects also showing above normal CK activity. Although the time-course response of CK-MB activity was virtually identical to that of total CK activity, only the 6-hour and 12-hour intervals were significantly higher than pre-exercise activity (Figure 2). As was the case with total CK activity, mean values showed a steady increase in activity with before 0, 0 to 6, and 6 to 12 hour increases of 11%, 15%, and 11%, respectively. The cumulative effect of these between-interval increases resulted in a peak elevation of 43% between pre-exercise and 12 hours. CK-MB reached a maximum value of 13.0 +_ 1.51 U/L at 12 hours and was followed by a steady decrease over the next 36 hours. At 48 and 72 hours, activity reached 9.0 + 0.44 U/L and 9.0 + 0.38 U/L, respectively. Subsequent to the apparent plateau in activity between 48 and 72 hours postexercise, CK-MB activity increased in a fashion similar to that observed with total CK activity. Fifty-two percent (11 of 21) of subjects showed an increase in CK-MB activity between the 72- and 96-hour intervals with a mean increase

HAYWARD ET AL • AMI INDICATORS FOLLOWING ECCENTRIC EXERCISE

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of 4.4 U/L. Of those individuals that showed an increase in total CK between 72 and 96 hours, 83% (10 of 12) also showed a corresponding increase in CK-MB at these data collection periods. The range of values considered normal for CK-MB varies. Cutoff values for the upper limit of normal have been reported as 10 U/L, 2°,21 13 U/L, 22 and 24 U / L ? 3 When using 10 U/L as the upper limit of normal, 28% (47 of 168) of the CK-MB assays show above-normal values. Additionally, 57% (12 of 21) of subjects exceeded this cutoff value during at least one data collection interval. Increasing the cutoff value to 13 U/L decreased the occurrence of above-normal presentations to 11% (19 of 168) of assays and 38% (8 of 21) of subjects. Using the most conservative cutoff value of 24 U/L, the percentages decreased further to 5% of assays (9 of 168) and 24% of subjects (5 of 21) showing abnormal values.

Leukocyte Response All participants showed pre-exercise values for total circulating leukocytes within the normal range for healthy adult men. Cell counts were significantly increased over pre-exercise values at the 0, 6, and 12 hour intervals (Figure 3). The exercise intervention resulted in a transient increase in total leukocytes, with the largest increase occurring immediately after the exercise bout, resulting in a 21% elevation over pre-exercise levels. Leukocyte count continued to increase at 6 hours postexercise, reaching a peak value of 8.12 _+ 0.37 X 10 9 cells/L at 12 hours. This amounted to a peak increase in total leukocytes of 36%. By 24 hours postexercise total circulating leukocytes had dropped to pre-exercise levels and remained there throughout the duration of the data collection period. Observations of individual data showed that 86% (18 of 21) of the subjects showed an increase in circulating leukocytes at 0 hours, with the remainder (3 of 21) showing a slight decrease. Additionally, all subjects showed elevated total leukocyte counts at some point during the first 12 hours postexercise. As for the lymphocyte count, significant increases were observed at 0 and 12 hours postexercise (Figure 4). Immediately after the exercise lymphocytes showed a 25% increase,

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FIGURE 3. Total leukocyte response to the exercise bout. Values are mean + SEM. The solid box indicates the exercise intervention. *Significantly different from pre-exercise values. rising from a pre-exercise level of 2.28 + 0.13 × 10 9 cells/L (38% of leukocytes) to 2.84 + 0.24 × 109 cells/L (39% of leukocytes) at 0 hours. This was followed by a decrease to 2.31 + 0.15 × 109 cells/L (31% of leukocytes) at 6 hours, resulting in a 19% decrease from the previous interval. At 12 hours lymphocytes had again increased to 2.50 _+ 0.13 × 109 cells/L (31% of leukocytes), which equates to a 8.5% increase from 6 hours. Lymphocyte values then remained relatively constant throughout the balance of the investigation, ranging from 2.18 + 0.14 × 109 cells/L (36% of leukocytes) to 2.08 -+ 0.11 × 109 cells/L (34% of leukocytes). Of note is the relatively large variability displayed at 0 hours postexercise. The mean range for all others within time intervals (6, 12, 24, 48, 72, and 96 hours) was calculated to be 1.70 × 109 cells/L as opposed to a range of 4.00 × 109 cells/L at 0 hours. This is also seen in the larger standard error at this time period, which is substantially higher than at any other time period. Although the maximum value for a single subject at 0 hours was 5.39 × 10 9 cells/L, only 14% (3 of 21) of subjects presented values exceeding 3.5 X 109 cells/L. At 0 hours 81% (17 of 21) of subjects showed increases in circulating lymphocytes. Of the 3 subjects not showing such an increase, 2 were the same 3.2 7

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subjects showing a decrease in total leukocytes at the same time interval.

TABLE 1.

Variable Interrelationship After the exercise intervention, both mean total CK activity as well as mean total leukocyte count increased over time in a similar fashion. To determine the relationship between these two variables, a simple linear regression was conducted. The analysis yielded a correlation coefficient of r = .273 (P < .001). However, this correlation also resulted in an r 2 = .075 with only 7.5% of the variance in total CK activity explained by total leukocyte count. A similar analysis was conducted using CK-MB activity as the dependent variable while maintaining total leukocyte count as the independent variable. The correlation coefficient between the variables was determined to be r = .085 (P = .287). In this analysis the total leukocyte count accounted for virtually none of the variance observed in CK-MB activity (0.7%). Observation of individual data showed considerable variation in the time-course response for total CK activity, CK-MB activity, as well as total leukocyte count (individual data not presented). Thus, to account for such individual differences, a simple linear regression was conducted using peak CK activity and peak total leukocyte count as well as a separate analysis using peak CK-MB activity and peak total leukocyte count. Analysis of peak CK activity and peak leukocyte count yielded a somewhat higher correlation (r = .427); however, the correlation coefficient was determined to be above the level of significance (P = .061). Using this analysis, peak leukocyte values accounted for just over 18% of the variability observed in peak CK activity. Likewise, the corresponding CK-MB analysis showed similar alterations when peak values were used as opposed to all possible values. In this case the correlation coefficient was determined to be r = .208 (P = .379). Although this analysis yielded a higher correlation coefficient, little variance in CK-MB activity (4.3%) was explained by total leukocyte count. There are a number of biochemical standards used in the determination of AMI. Of these, the most common standards use some measure of CK-MB, whereas others use total CK activity in conjunction with CK-MB measures. The data from this investigation were compared using five standards, which are described in Table 1. As expected, there exists an incremental decrease in the occurrence of values that exceed AMI-positive cutoff values as the criterion become more conservative. Using criterion A, 21% (36 of 168) of the data points meet the criterion for positive AMI determination (Figure 5). These 36 data collection points occurred in 13 of the 21 subjects (61%) and appeared in at least 3 subjects at each of the eight data collection intervals. As for criterion B, only 63 data collection points could be used in the analysis because the final four intervals were separated by more than 12 hours (Table 1). However, 11% (7 of 63) of the data collection points did show elevations above the lower limit for the detection of AMI. These seven data collection points occurred at least one time at all possible data collection intervals (0, 6, and 12 hours) and were shown by 6 of the 21

Criteria Used in Determination of AMI Total CK

CK-MB

Comment

Criterion A

>10 U/L

CK-MB > 5% of total CK

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->13 U/L

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CK-MB > 24 U/L

CK-MB 5.5-20% of total CK

Criterion C

>191 U/L

Criterion D

Relative lymphocyte percentage <20.3% of total leukocytes

Criterion E

---13 U/L

Relative lymphocyte percentage <20.3% of total leukocytes

subjects (29%). With the most conservative criterion, criterion C, the occurrence of AMI-positive data collection points decreased to 5% (9 of 168). These nine data collection points were limited to the 6, 12, 24, and 96 hour intervals and were presented by 24% (5 of 21) of subjects. Criterion D suggests that a relative lymphocyte percentage that accounts for less than 20.3% of the total leukocyte count is a strong independent predictor of AMI. 16 A comparison of the absolute leukocyte count and the absolute iymphocyte count shows that of the 168 data points only 3 (1.8%) meet this standard (Figure 6). The three data collection points were presented by three different subjects (14%, 3 of 21) at three different data collection intervals (6, 12, and 24 hours). Criterion E presents the use of relative lymphocyte count in conjunction with CK-MB activity as indicators of AMI. For this comparison, cutoff value for relative lymphocyte percentage is 20.3% with cutoff values for CK-MB activity set at 13 U/L. Results show that although three (1.8%) data collection points fall below the cutoff value for relative lymphocyte percentage and 19 (11%) data collection points exceed the upper limit of normal for CK-MB activity, no data collection point meets both criteria (0%). Thus when used in combination, relative lymphocyte percentage and CK-MB activity yielded no AMI-positive results (Figure 7). 25 I

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DISCUSSION Our results appear to be among the minority of studies that show peak CK activity at less than 24 hours postexercise. There appear to be no common characteristics associated with studies observing peak values either before the 24-hour interval or after the 24-hour interval. As for those investigations using bench stepping as the mode of exercise, peak activity has been reported to be at 17 or beyond 18 the 24-hour interval. Postexercise absolute values for total CK activity were relatively low in comparison to the contemporary literature, with peak mean activity reaching only 208 +_ 17.4 U/L. Other investigations surrounding exerciseinduced muscle injury report values in excess of 10,000 U/L, a4 with bench stepping exercises eliciting values exceeding 8,000 U/L l° and 34,500 U/L 18 in selected subjects. The present results are not in agreement with the contention proposed by Clarkson et al I regarding high, medium, and low responders. However, this grouped response appears to have substantial merit and has been observed in numerous investigations, including investigations in our laboratory. 9,1° A consistent finding in the literature is the highly individualized response of total CK activity to eccentric exercise. This finding was also observed in our investigation and is 50-

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111

supported by the observance of relatively large standard error values (12 hours, 17.4; 96 hours, 26.2). The comparatively low values obtained in this investigation were a desired outcome by the investigators. This was because the protocols used in a majority of the literature use an exercise modality that is novel to the participants and the activity is sustained for an extended period of time. Thus the results fail to apply to typical daily (weekly/monthly) experiences. The exercise modality selected for this investigation used major lower-limb muscle groups for a relatively short period of time. The goal was to select an exercise mode that may be encountered by a given individual and to sustain that exercise for a moderate period of time. There appear to be two viable explanations for the observation of relatively low total CK activity. First, the relatively low activity may be because all subjects are what have been termed low responders. With the exception of one subject at only one time interval (538 U/L at 96 hours), all data collection points were below the upper limit of what has been identified as a low responder (500 U/L). A second explanation may be that the exercise intensity and duration were moderate enough to induce only modest increases in total activity. Of these two factors, the duration of the exercise (20 minutes) may be of most significance. Few investigations have observed the response of CK-MB activity after exercise, and the majority of those that have observed its response primarily focused their attention on highly trained marathon runners. Thus, no substantial comparisons can be made to the results obtained from the present investigation. The CK-MB isoenzyme has been reported to constitute approximately 1% of total CK activity in skeletal muscle. Therefore increases in CK-MB that are disproportional to its relative percentage are unexpected. The results from the present investigation show that CK-MB activity can significantly increase even after moderate exercise. Although CK-MB activity increased significantly at 6 and 12 hours postexercise, the relative percentage of the MB fraction accounted for only 6.9% and 6.3% of the total CK activity. In fact, the highest relative percentage of the MB fraction was obtained at the pre-exercise interval, which accounted for 7.8% of the total activity. All intervals ranged from a high of 7.8% (before) to a low of 5.7% (24 and 96 hours). Although the relative percentage of CK-MB activity obtained from mean values appears to be somewhat stable and within the expected range, the most pronounced observations are seen in the analysis of individual data. As previously discussed, there is a great deal of ambiguity concerning standards with which normal and abnormal results can be determined. As the upper limit of normal is increased, there will obviously be fewer occurrences of above-normal results. However, using even the most conservative standards (-->24 U/L), the present investigation showed that selected individuals may still present activity values that exceed the upper limit of normal. The observance of selected individuals (non-endurance-trained) presenting such a response has been reported elsewhere,9,10, 25 and is a recognized concern for the emergency room physician. 12,26 Conflicting results have been reported for leukocyte response to exercise, and even greater confusion has been reported after an exercise bout designed to elicit skeletal muscle injury.27,2s It appears that the primary conflict is

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AMERICAN JOURNAL OF EMERGENCY MEDICINE [] Volume 16, Number 2 [] March 1998

caused by a lack of standardization among the research investigations. Each investigation measures variables at differing intervals, which does not allow for an exact determination of when, for example, peak values are reached. The observation that leukocytosis occurs at some point after an exercise bout is consistent among all studies. Piazza et a127 reported that after 60 minutes of downhill running, peak leukocyte count was obtained immediately after the exercise bout. The results presented here show a much different response, with leukocyte counts increasing steadily, peaking at 12 hours postexercise, and returning to resting levels by 24 hours. As for the degree of response, it appears as though the subjects in this investigation experienced a moderate leukocytosis. Exercise of high intensity has been reported to result in increases approximating 100%, 29,3o whereas lowintensity exercise results in increases closer to 25%. 32 In this experiment, bench-stepping exercise resulted in a leukocytosis of 36%. Lymphocyte values obtained from this study appear to be in agreement with what now appears to be the standard biphasic alteration as a result of exercise regardless of the exercise intensity. The results showed an initial increase in lymphocyte count followed by a return to near resting values 6 hours postexercise and a secondary increase at 12 hours. Pizza et a132 reported a significant (P < .05) decrease in lymphocytes 1.5 hours postexercise in comparison to preexercise values, with near resting values regained by 12 hours. Thus, it appears as though lymphocyte values could have decreased even further between the 0 and 6 hours data collection intervals, yet remained undetected because of the sampling times. Nevertheless, these results appear to be in agreement with other investigations showing peak lymphocyte values immediately postexercise. 31,33,34 Visual observation of the responses of total leukocytes and total CK appears suggestive of some degree of relationship because of the fact that both follow a relatively similar time course. However, regression analysis proved otherwise with an r 2 = .075. A subsequent regression was conducted using only those values obtained during the first four data collection intervals, because the similarity in time-course appeared to dissipate after the 12-hour interval. This analysis yielded virtually identical results with and r 2 -- .076 (data not presented), further substantiating the absence of a significant relationship between these two variables. Similar results were obtained from analyses comparing CK-MB versus leukocytes (r 2 = .007), peak CK versus peak leukocytes (r 2 = . 182), and peak CK-MB versus peak leukocytes (r 2 = .043). Few investigations have analyzed data in this fashion, and therefore comparisons are difficult. However, the present results do suggest the lack of a significant positive or negative relationship between any of the aforementioned variables. This is not surprising because circulating leukocytes are primarily under nervous and hormonal control, whereas the CK response is under the control of localized myocellular factors. At the core of this investigation is the response of serum indicators of AMI to exercise induced-muscle injury. Again, it should be pointed out that observations of mean values obtained on all observed parameters remain within the expected range. It is only on the analysis of individual data that deviations from the expected values are observed. In the

clinical setting, multiple factors are used in the determination of AMI, some of which include biochemical alterations in blood parameters. Although numerous parameters have been proposed as independent indicators of AMI, CK-MB activity is one of the most widely used. Several differing criteria surrounding CK-MB may be used in the determination of an AMI. Using CK-MB activity alone yields a substantial number of AMI-positive cases regardless of the criteria selected. Using even the most conservative CK-MB activity criterion (->24 U/L), 5% (9 of 168) of the data collection points meet the AMI-positive standard. This may not seem to be a considerable number; however, the magnitude of such an observation is realized when it is considered that the AMI-positive criteria for CK-MB occurred in 24% (5 of 21) of the experimental subjects during at least one data collection interval. The interpretation of such a finding is that if any of these individuals experience angina in the absence of AMI and pursue admission into an emergency care system, the risk of a false-positive AMI diagnosis is greatly enhanced. It has recently been proposed that the use of the relative lymphocyte percentage is an independent indicator of AMI. The value of using such an indicator is that the leukocyte differential count is relatively inexpensive, and is conducted routinely for individuals presenting with chest pain. The underlying mechanism associated with the use of the leukocyte differential as an indicator of AMI involves the integration of the nervous, endocrine, and immune systems. Myocardial afferent nerves signal information to the central nervous system regarding ischemic conditions and possible myocardial damage. 35The hypothalamus then releases ACTH as a result of the normal stress response, which in turn stimulates the release of cortisol from the adrenal cortex. Cortisol results in the modification of the leukocyte differential, 36-39partly by inducing a relative lymphocytopenia. 4°,41 It is suggested that a relative lymphocytopenia below 20.3% can function as a sensitive and specific indicator of AMI. 16 Using this standard, the present results showed that only three (18.9%, 20.0%, 20.1%) data collection points showed such a response. No pattern could be detected regarding the time interval of lymphocytopenia, with positive values obtained at three separate intervals. Thomson et a116 suggested that the combination of both a relative lymphocytopenia and an elevated CK-MB mass was an accurate marker of AMI that improves the sensitivity of diagnosis compared with ECG changes (ST-segment) alone. This may be considered the primary limitation to our investigation because Thomson et al ~6 used CK-MB mass and the CK-MB mass assay is also widely used in the clinical setting, whereas this study used CK-MB activity. The data obtained from this investigation clearly support the use of both CK-MB as well as relative lymphocyte percentage, at least as far as false-positive results caused by complications associated with exercise are concerned. Alone, both the CK-MB activity and leukocyte differential count did present individuals who exceeded the limits suggestive of AMI. However, the implementation of both techniques in combination completely eliminated AMI-positive biochemical results in our subjects. Other investigations have reported peak lymphocytopenia within the first 1.5 hours postexerciseY ,31'34 a time interval

HAYWARD ET AL [] AMI INDICATORS FOLLOWING ECCENTRIC EXERCISE

not monitored in this investigation. If that time interval had been monitored, the results may have presented a higher incidence of AMI-positive values for the leukocyte differential analysis. However, even if that time period was monitored, the likelihood of observing data points positive for both the CK-MB activity and leukocyte differential would not increase substantially because CK-MB activity peaked at 12 hours postexercise. It is therefore suggested that the value of these tests in combination is not affected by the time at which the sample is obtained. It appears as though the future of early AMI assessment is headed in the direction of cardiac-specific contractile protein determination, such as troponin I, troponin T, and myosin light chain 1. However, until these methodologies become readily available in the clinical setting, the use of CK-MB activity in conjunction with leukocyte and leukocyte snbpopulation count may provide incremental value in the detection of AMI as well as reduce the incidence of misdiagnosis associated with exercise.

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