Specificity of Elevated Serum MB Creatine Phosphokinase Activity in the Diagnosis of Acute Myocardial Infarction
ROBERT ROBERTS, MD K. S. GOWDA, MD PHILIP A. LUDBROOK, MD BURTON E. SOBEL, MD, FACC St. Louis, Missouri
From the Cardiovascular Division, Washington University, St. Louis, Mo. This investigation was supported in part by U. S. Public Health Service Contract 7%2481 and U. S. Public Health Service Grant HL 16705. Manuscript accepted January 15, 1975. Address for reprints: Burton E. Sobel, MD, Cardiovascular Division, Washington University, 660 South Euclid, St. Louis, Mo. 63110.
Creatine phosphokinase (CPK) isoenzyme determinations are useful in the diagnosis of myocardial infarction. However, until suitably sensitive and precise quantitative procedures became available, the diagnostic specificity of serum CPK isoenzyme elevations could not be thoroughly examined. In this study an assay procedure capable of accurately determining activity of individual CPK isoenzymes even in serum samples with normal total CPK activity was employed to obtain two types of information. First, CPK isoenzyme profiles were examined in extracts of a spectrum of human tissues obtained at operation to determine whether the isoenzyme associated with myocardium is present in other human tissues in quantities sufficient to produce increased activity in serum. In addition, CPK isoenzymes were analyzed quantitatively in serial serum samples from 50 hospitalized control subjects, 100 patients with acute myocardial infarction, 100 patients undergoing noncardiac surgery and 50 patients undergoing cardiac catheterization to determine whether insult to tissues othe~ than the heart is associated with increased "myocardial" CPK isoenzyme activity in serum. Results from analyses of tissue extracts indicated that myocardium is the only tissue surveyed containing sufficient MB CPK to account for substantial increases in serum MB activity. Results from analyses of serial serum samples indicated that MB CPK activity levels are consistently elevated after myocardial infarction, averaging 0.089 IU/ml. However, after cardiac catheterization or noncardiac surgery peak serum MB activity remains low, averaging only 0.004 IU/ml despite marked elevations in total serum CPK activity. Thus, elevated serum MB CPK activity is a highly specific as well as sensitive criterion of myocardial injury.
Soon after the pioneering observations by Karmen et al., 1 increased activity of selected serum enzymes became the established hallmark of acute myocardial infarction. Elevation of serum creatine phosphokinase levels (CPK) appears to be the most sensitive conventional serum enzyme criterion of acute myocardial infarction,2-4 but false positive results occur with a frequency of approximately 15 percent. 3 Recent technical advances in CPK isoenzyme analysis led to the observation that increased activity of serum MB CPK (an isoenzyme associated with myocardium) is a useful index of acute myocardial infarction. 5,6 We 7 have shown that increased serum CPK activity after intramuscular injections is not associated with elevated MB CPK levels in contrast to the case after acute myocardial infarction, but the overall specificity of elevated serum MB CPK activity as a diagnostic criterion of myocardial infarction remains to be thoroughly elucidated. In this study we sought to determine whether human tissue outside the heart contained sufficient amounts of MB CPK to account for appreciable elevations of MB CPK activity in serum. We have recently
October 6, 1975
The American Journal of CARDIOLOGY
Volume 36
433
SERUM MB CPK IN MYOCARDIAL INFARCTION--ROBERTS ET AL.
developed a quantitative p r o c e d u r e for assay of activity of individual C P K isoenzymes, with a sensitivity of 0.002 IU/ml and reproducibility of +2 percent, s T h i s technique, which avoids some potentially serious quantitative difficulties associated with conventional methods, was employed in this s t u d y to assay C P K isoenzymes in extracts of h u m a n tissues obtained freshly during surgery from the myocardium, skeletal muscle, brain, stomach, colon, small intestines, lung, spleen, liver and kidney and washed red blood cells. Although some estimates of C P K isoenzyme activity have been m a d e previously in tissues o b t a i n e d at necropsy, a systematic evaluation of C P K isoenzyme profiles in freshly obtained h u m a n tissues employing a quantitative assay procedure has not been available. A second goal of this s t u d y was to d e t e r m i n e whether insult to tissues other t h a n the h e a r t produced serum MB C P K changes simulating those seen in patients with acute myocardial infarction. Accordingly, C P K isoenzymes were assayed in serial serum samples from 100 patients with definite myocardial infarction, 100 patients undergoing thoracic, abdominal, orthopedic or genitourinary surgical procedures, 50 patients undergoing cardiac catheterization and 50 hospitalized control subjects. Results of analyses of h u m a n tissue extracts obtained at operation and of serial serum samples in these patients indicate t h a t m y 0 c a r d i u m is the only h u m a n tissue containing sufficient M B C P K to account for substantial elevations of M B C P K levels in serum. T h e y confirm previous observations indicating t h a t consistent elevations of serum MB C P K activity a c c o m p a n y acute myocardial infarction 5,6 and t h e y d e m o n s t r a t e t h a t surgical t r a u m a to organs other t h a n the h e a r t does not produce elevated serum MB C P K activity even when total C P K activity is m a r k e d l y increased. 9 M a t e r i a l s and M e t h o d s Tissue and Serum Sampling Procedures
Fresh samples of human skeletal muscle (deltoid, pectoralis major, vastus media lis and rectus abdominis), myocardium, stomach, small intestines, colon, brain, lung, liver, spleen and kidney were obtained from patients at the time of surgery required for therapy or diagnosis. Samples were minced, suspended in 25 ml/g of homogenizing medium and homogenized in 0.25 molar sucrose; 0.01 molar Tris, pH 7.4; 0.001 molar ethyleneglycoltetraacetic acid (EGTA), pH 7.4; and 0.001 molar mercaptoethanol. Blood samples were collected in 0.005 molar neutralized EGTA, centrifuged at 2,000 g for 10 minutes and decanted. Mercaptoethanol (0.005 molar) was added to avoid loss of CPK activity. Patients Studied
Patients with acute myocardial infarction: Blood samples were obtained serially from 100 patients admitted to the Barnes Hospital Cardiac Care Unit with definite transmural myocardial infarction manifested by a typical history of chest pain, serial electrocardiographic changes with development of Q waves and characteristic serial changes in serum transaminase and lactic dehydrogenase (LDH). Blood samples were collected every 2 hours
434
October 6, 1975
The American Journal of CARDIOLOGY
through a peripheral venous indwelling cannula for a total of 48 hours. Samples were assayed immediately for CpK isoenzyme activity or fast-frozen, stored at - 2 0 ° C and assayed within 4 weeks. Samples stored under these conditions do not exhibit loss of activity for at least 6 weeks, s Hospitalized control subjects; Samples were obtained in the same fashion from 50 patients admitted because of suspected but unconfirmed acute myocardial infarction who served as control subjects. Patients undergoing noncardiac surgery: One hundred patients undergoing surgical procedures (Table I) were studied. None exhibited clinical or electrocardiographic evidence of myocardial infarction during hospitalization. Blood samples were obtained immediately before operation, as soon as possible offer operation, and every 6 hours thereafter for 24 hours. Patients undergoing cardiac catheterization: Fifty patients were studied after combined right and left heart catheterization with Selective coronary arteriography and ventricul0graphy. The studies were performed in 40 patients because of suspected coronary artery disease and in 10 because of valve disease. Coronary arteriography was performed by the Sones technique (36 cases) or the Judkins technique (14 cases). Before catheterization patients were medicated with pentobarbital sodium (Nembutal®), 100 mg, and diazepam (Valium®), 5 mg, given intramuscularly. Transitory chest pain occurred in 10 patients during catheterization, but no patient studied exhibited clinical or electrocardiographic evidence of definite myocardial infarction. Blood samples for CPK determinations were obtained before and after catheterization and at 2 hour intervals subsequently for 24 hours. Biochemical Determinations
Total CPK activity was determined spectrophotometrically7 in serum samples and in 50 td aliquots of tissue extracts diluted with Tris, 0.01 molar, pH 7.4, and bovine serum albumin, 2 mg/ml, so that CPK activity was within the range of linearity of the assay. Samples were assayed with and without creatine phosphate as substrate to exclude contributions to apparent activity from myokinase and other moieties. CPK isoenzymes were assayed quantitatively with the kinetic fluorometric procedure recently described, s Results with this procedure are linear with respect to time and enzyme activity and reproducible within 4-2 percent. As little as 0.002 IU/ml of each isoenzyme can be detected under conditions that avoid several difficulties associated with quantification of isoenzyme activity by fluorescence scanning of electrophoretic media. As reported previously, serum samples from normal subjects assayed under these conditions contain less than 0.004 IU/ml of MB CPK. Results CPK Isoenzyme Activity in Tissue Extracts
Total C P K activity and C P K isoenzyme activity in extracts of h u m a n skeletal muscle, heart, brain, stomach, small intestines, colon, liver, spleen, lung, kidney and red blood cells are shown in Figure 1. As c a n be seen, C P K activity (expressed as IU/g wet weight of tissue) is greatest in skeletal muscle, exceeding activity in m y o c a r d i u m b y more t h a n 300 percent. Substantial a m o u n t s of C P K activity occur only in skeletal muscle, heart, brain and gastrointestinal tract. C P K activity in lung and k i d n e y is less t h a n 3 p e r c e n t of t h a t in myocardium.
Volume 36
SERUM MB CPK IN MYOCARDIAL INFARCTION--ROBERTS ET AL.
TABLE I
TABLE II
Noncardiac Surgical Procedures in Patients Studied
Seru m CPK and MB CPK Activity in Patients
Procedure Thoracic Pneumonectomy Exploratory thoracotomy Hiatus hernia repair Abdominal Gastrectomy Splenectomy Intestinal bypass Colectomy Genitourinary Prostatectomy Hysterectomy Orthopedic Total hip replacement Meniscectomy Tendon repair Total
Patients (no.) Category 20 5 5
Hospitalized controls Acute myocardial infarction Post-cardiac catheterization Post-surgery Thoracic Abdominal Genitourinary Orthopedi c
20 5 6 7 16 6 4 3 3 100
Mean Peak Total CPK Activity (!U/ml)
Mean Peak MB CPK Activity (IU/ml!
50
0.038 -+ 0.011
0.002 -+ 0.001
100
0.852 -+ 0.120
0.089 +- 0.032
50
0.260 -+ 0.033
0.004 -+ 0.001
0.850 1.260 0.760 0.842 0.540
0.003 0.004 0.003 0.002 0.003
Patients (no.)
100 30 38 22 10
+- 0.260 -+ 0.320 -+ 0.106 +- 0.220 + 0.180
-+ 0,001 +_0.001 -+ 0.00t ,-+0.0Q1 -~ 0.001
Results are expressed as means _+standard deviation. Skeletal Muscle
Of the tissues surveyed, only myocardium contains significant amounts of MB CPK. Under the assay conditions used, only the MM isoenzyme was detectable in extracts of skeletal muscle.
Serum CPK Isoenzymes Control hospitalized subjects: As previously reported with a smaller group of patients, s CPK activity in serum samples from hospitalized patients with suspected but unconfirmed acute myocardial infarction averaged 0.038 ± 0.011 (standard deviation) and MB CPK activity averaged 0.002 ± 0.001 IU/mi (Table II). No hospitalized control subject exhibited MB CPK activity exceeding 0.005 IU/ml. Patients with myocardial infarction: All 100 patients with definite myocardial infarction exhibited elevated serum levels of total CPK activity. Peak activity averaged 0.852 ± 0.120 IU/ml (Table II). The mean interval between the initial elevation of serum C P K activity and the time of occurrence of peak activity was 15.2 ± 3 hours (range 10 to 18 hours). The interval from the time of onset of chest pain to the time when CPK activity first exceeded the normal range was 4 ± 2 hours. Elevated MB CPK activity was observed in all patients with acute myocardial infarction. Peak MB CPK activity averaged 0.089 ± 0.032 tU/ml (range 0.025 to 0.215). MB CPK activity was generally detectable by fluorescence scanning within 4 hours after the onset of chest pain, when total CPK activity had increased by 50 to 100 percent above control values. Modestly increased MB C P K activity was detected slightly earlier with the quantitative technique, often within 3 hours after the onset of chest pain. Peak MB and peak total CPK activity occurred at about the same time. Patients undergoing noncardiac surgery: All patients in this group exhibited elevated serum levels of total CPK activity (Table II). However, MB CPK elevations were not detected in any of the serial samples from these patients. Only M M CPK was detectable by electrophoresis and, with the quantitative assay technique, only minimal increases in serum MB
3200
.?
~
Heart
[ ] Total CPK
600 50o
[ ] MM CPK t
[ ] BB CPK
§
•
M8 CPK
D
~Z 400 >I---
~. 300 p-
u
ROBERT ROBERTS, MD K. S. GOWDA, MD PHILIP A. LUDBROOK, MD BURTON E. SOBEL, MD, FACC St. Louis, Missouri
From the Cardiovascular Division, Washington University, St. Louis, Mo. This investigation was supported in part by U. S. Public Health Service Contract 7%2481 and U. S. Public Health Service Grant HL 16705. Manuscript accepted January 15, 1975. Address for reprints: Burton E. Sobel, MD, Cardiovascular Division, Washington University, 660 South Euclid, St. Louis, Mo. 63110.
Creatine phosphokinase (CPK) isoenzyme determinations are useful in the diagnosis of myocardial infarction. However, until suitably sensitive and precise quantitative procedures became available, the diagnostic specificity of serum CPK isoenzyme elevations could not be thoroughly examined. In this study an assay procedure capable of accurately determining activity of individual CPK isoenzymes even in serum samples with normal total CPK activity was employed to obtain two types of information. First, CPK isoenzyme profiles were examined in extracts of a spectrum of human tissues obtained at operation to determine whether the isoenzyme associated with myocardium is present in other human tissues in quantities sufficient to produce increased activity in serum. In addition, CPK isoenzymes were analyzed quantitatively in serial serum samples from 50 hospitalized control subjects, 100 patients with acute myocardial infarction, 100 patients undergoing noncardiac surgery and 50 patients undergoing cardiac catheterization to determine whether insult to tissues othe~ than the heart is associated with increased "myocardial" CPK isoenzyme activity in serum. Results from analyses of tissue extracts indicated that myocardium is the only tissue surveyed containing sufficient MB CPK to account for substantial increases in serum MB activity. Results from analyses of serial serum samples indicated that MB CPK activity levels are consistently elevated after myocardial infarction, averaging 0.089 IU/ml. However, after cardiac catheterization or noncardiac surgery peak serum MB activity remains low, averaging only 0.004 IU/ml despite marked elevations in total serum CPK activity. Thus, elevated serum MB CPK activity is a highly specific as well as sensitive criterion of myocardial injury.
Soon after the pioneering observations by Karmen et al., 1 increased activity of selected serum enzymes became the established hallmark of acute myocardial infarction. Elevation of serum creatine phosphokinase levels (CPK) appears to be the most sensitive conventional serum enzyme criterion of acute myocardial infarction,2-4 but false positive results occur with a frequency of approximately 15 percent. 3 Recent technical advances in CPK isoenzyme analysis led to the observation that increased activity of serum MB CPK (an isoenzyme associated with myocardium) is a useful index of acute myocardial infarction. 5,6 We 7 have shown that increased serum CPK activity after intramuscular injections is not associated with elevated MB CPK levels in contrast to the case after acute myocardial infarction, but the overall specificity of elevated serum MB CPK activity as a diagnostic criterion of myocardial infarction remains to be thoroughly elucidated. In this study we sought to determine whether human tissue outside the heart contained sufficient amounts of MB CPK to account for appreciable elevations of MB CPK activity in serum. We have recently
October 6, 1975
The American Journal of CARDIOLOGY
Volume 36
433
SERUM MB CPK IN MYOCARDIAL INFARCTION--ROBERTS ET AL.
developed a quantitative p r o c e d u r e for assay of activity of individual C P K isoenzymes, with a sensitivity of 0.002 IU/ml and reproducibility of +2 percent, s T h i s technique, which avoids some potentially serious quantitative difficulties associated with conventional methods, was employed in this s t u d y to assay C P K isoenzymes in extracts of h u m a n tissues obtained freshly during surgery from the myocardium, skeletal muscle, brain, stomach, colon, small intestines, lung, spleen, liver and kidney and washed red blood cells. Although some estimates of C P K isoenzyme activity have been m a d e previously in tissues o b t a i n e d at necropsy, a systematic evaluation of C P K isoenzyme profiles in freshly obtained h u m a n tissues employing a quantitative assay procedure has not been available. A second goal of this s t u d y was to d e t e r m i n e whether insult to tissues other t h a n the h e a r t produced serum MB C P K changes simulating those seen in patients with acute myocardial infarction. Accordingly, C P K isoenzymes were assayed in serial serum samples from 100 patients with definite myocardial infarction, 100 patients undergoing thoracic, abdominal, orthopedic or genitourinary surgical procedures, 50 patients undergoing cardiac catheterization and 50 hospitalized control subjects. Results of analyses of h u m a n tissue extracts obtained at operation and of serial serum samples in these patients indicate t h a t m y 0 c a r d i u m is the only h u m a n tissue containing sufficient M B C P K to account for substantial elevations of M B C P K levels in serum. T h e y confirm previous observations indicating t h a t consistent elevations of serum MB C P K activity a c c o m p a n y acute myocardial infarction 5,6 and t h e y d e m o n s t r a t e t h a t surgical t r a u m a to organs other t h a n the h e a r t does not produce elevated serum MB C P K activity even when total C P K activity is m a r k e d l y increased. 9 M a t e r i a l s and M e t h o d s Tissue and Serum Sampling Procedures
Fresh samples of human skeletal muscle (deltoid, pectoralis major, vastus media lis and rectus abdominis), myocardium, stomach, small intestines, colon, brain, lung, liver, spleen and kidney were obtained from patients at the time of surgery required for therapy or diagnosis. Samples were minced, suspended in 25 ml/g of homogenizing medium and homogenized in 0.25 molar sucrose; 0.01 molar Tris, pH 7.4; 0.001 molar ethyleneglycoltetraacetic acid (EGTA), pH 7.4; and 0.001 molar mercaptoethanol. Blood samples were collected in 0.005 molar neutralized EGTA, centrifuged at 2,000 g for 10 minutes and decanted. Mercaptoethanol (0.005 molar) was added to avoid loss of CPK activity. Patients Studied
Patients with acute myocardial infarction: Blood samples were obtained serially from 100 patients admitted to the Barnes Hospital Cardiac Care Unit with definite transmural myocardial infarction manifested by a typical history of chest pain, serial electrocardiographic changes with development of Q waves and characteristic serial changes in serum transaminase and lactic dehydrogenase (LDH). Blood samples were collected every 2 hours
434
October 6, 1975
The American Journal of CARDIOLOGY
through a peripheral venous indwelling cannula for a total of 48 hours. Samples were assayed immediately for CpK isoenzyme activity or fast-frozen, stored at - 2 0 ° C and assayed within 4 weeks. Samples stored under these conditions do not exhibit loss of activity for at least 6 weeks, s Hospitalized control subjects; Samples were obtained in the same fashion from 50 patients admitted because of suspected but unconfirmed acute myocardial infarction who served as control subjects. Patients undergoing noncardiac surgery: One hundred patients undergoing surgical procedures (Table I) were studied. None exhibited clinical or electrocardiographic evidence of myocardial infarction during hospitalization. Blood samples were obtained immediately before operation, as soon as possible offer operation, and every 6 hours thereafter for 24 hours. Patients undergoing cardiac catheterization: Fifty patients were studied after combined right and left heart catheterization with Selective coronary arteriography and ventricul0graphy. The studies were performed in 40 patients because of suspected coronary artery disease and in 10 because of valve disease. Coronary arteriography was performed by the Sones technique (36 cases) or the Judkins technique (14 cases). Before catheterization patients were medicated with pentobarbital sodium (Nembutal®), 100 mg, and diazepam (Valium®), 5 mg, given intramuscularly. Transitory chest pain occurred in 10 patients during catheterization, but no patient studied exhibited clinical or electrocardiographic evidence of definite myocardial infarction. Blood samples for CPK determinations were obtained before and after catheterization and at 2 hour intervals subsequently for 24 hours. Biochemical Determinations
Total CPK activity was determined spectrophotometrically7 in serum samples and in 50 td aliquots of tissue extracts diluted with Tris, 0.01 molar, pH 7.4, and bovine serum albumin, 2 mg/ml, so that CPK activity was within the range of linearity of the assay. Samples were assayed with and without creatine phosphate as substrate to exclude contributions to apparent activity from myokinase and other moieties. CPK isoenzymes were assayed quantitatively with the kinetic fluorometric procedure recently described, s Results with this procedure are linear with respect to time and enzyme activity and reproducible within 4-2 percent. As little as 0.002 IU/ml of each isoenzyme can be detected under conditions that avoid several difficulties associated with quantification of isoenzyme activity by fluorescence scanning of electrophoretic media. As reported previously, serum samples from normal subjects assayed under these conditions contain less than 0.004 IU/ml of MB CPK. Results CPK Isoenzyme Activity in Tissue Extracts
Total C P K activity and C P K isoenzyme activity in extracts of h u m a n skeletal muscle, heart, brain, stomach, small intestines, colon, liver, spleen, lung, kidney and red blood cells are shown in Figure 1. As c a n be seen, C P K activity (expressed as IU/g wet weight of tissue) is greatest in skeletal muscle, exceeding activity in m y o c a r d i u m b y more t h a n 300 percent. Substantial a m o u n t s of C P K activity occur only in skeletal muscle, heart, brain and gastrointestinal tract. C P K activity in lung and k i d n e y is less t h a n 3 p e r c e n t of t h a t in myocardium.
Volume 36
SERUM MB CPK IN MYOCARDIAL INFARCTION--ROBERTS ET AL.
TABLE I
TABLE II
Noncardiac Surgical Procedures in Patients Studied
Seru m CPK and MB CPK Activity in Patients
Procedure Thoracic Pneumonectomy Exploratory thoracotomy Hiatus hernia repair Abdominal Gastrectomy Splenectomy Intestinal bypass Colectomy Genitourinary Prostatectomy Hysterectomy Orthopedic Total hip replacement Meniscectomy Tendon repair Total
Patients (no.) Category 20 5 5
Hospitalized controls Acute myocardial infarction Post-cardiac catheterization Post-surgery Thoracic Abdominal Genitourinary Orthopedi c
20 5 6 7 16 6 4 3 3 100
Mean Peak Total CPK Activity (!U/ml)
Mean Peak MB CPK Activity (IU/ml!
50
0.038 -+ 0.011
0.002 -+ 0.001
100
0.852 -+ 0.120
0.089 +- 0.032
50
0.260 -+ 0.033
0.004 -+ 0.001
0.850 1.260 0.760 0.842 0.540
0.003 0.004 0.003 0.002 0.003
Patients (no.)
100 30 38 22 10
+- 0.260 -+ 0.320 -+ 0.106 +- 0.220 + 0.180
-+ 0,001 +_0.001 -+ 0.00t ,-+0.0Q1 -~ 0.001
Results are expressed as means _+standard deviation. Skeletal Muscle
Of the tissues surveyed, only myocardium contains significant amounts of MB CPK. Under the assay conditions used, only the MM isoenzyme was detectable in extracts of skeletal muscle.
Serum CPK Isoenzymes Control hospitalized subjects: As previously reported with a smaller group of patients, s CPK activity in serum samples from hospitalized patients with suspected but unconfirmed acute myocardial infarction averaged 0.038 ± 0.011 (standard deviation) and MB CPK activity averaged 0.002 ± 0.001 IU/mi (Table II). No hospitalized control subject exhibited MB CPK activity exceeding 0.005 IU/ml. Patients with myocardial infarction: All 100 patients with definite myocardial infarction exhibited elevated serum levels of total CPK activity. Peak activity averaged 0.852 ± 0.120 IU/ml (Table II). The mean interval between the initial elevation of serum C P K activity and the time of occurrence of peak activity was 15.2 ± 3 hours (range 10 to 18 hours). The interval from the time of onset of chest pain to the time when CPK activity first exceeded the normal range was 4 ± 2 hours. Elevated MB CPK activity was observed in all patients with acute myocardial infarction. Peak MB CPK activity averaged 0.089 ± 0.032 tU/ml (range 0.025 to 0.215). MB CPK activity was generally detectable by fluorescence scanning within 4 hours after the onset of chest pain, when total CPK activity had increased by 50 to 100 percent above control values. Modestly increased MB C P K activity was detected slightly earlier with the quantitative technique, often within 3 hours after the onset of chest pain. Peak MB and peak total CPK activity occurred at about the same time. Patients undergoing noncardiac surgery: All patients in this group exhibited elevated serum levels of total CPK activity (Table II). However, MB CPK elevations were not detected in any of the serial samples from these patients. Only M M CPK was detectable by electrophoresis and, with the quantitative assay technique, only minimal increases in serum MB
3200
.?
~
Heart
[ ] Total CPK
600 50o
[ ] MM CPK t
[ ] BB CPK
§
•
M8 CPK
D
~Z 400 >I---
~. 300 p-
u
