The creatine kinase system in the serum of runners following a doubling of training mileage

Summary This study examined the response of the serum creatine kinase (CK) isoenzyme/isoform system following the stress of successive weeks of increased training. Four highly-trained male long-distance runners doubled their training mileage [45.0 (SD 3.5) to 81.2 (4.7) miles] for 3 weeks. Venous blood samples were obtained prior to the start of the increased training period and at the end of each week, days 7, 14, and 21, 12 h after a training run. All runners experienced a significant increase in training perception difficulty, which correlated with the significant increase in training mileage. While total CK activities did not become significantly elevated until day 14, the tissue isoform CK-MM3, as well as the ratio of CK-MM3 to MM1 (final CK-MM degradation isoform) became significantly elevated at day 7 and remained elevated throughout day 21. These findings demonstrate that an increase in training mileage caused a significant and continuous release of tissue CK activity from skeletal muscle into the circulation.     

Measurement of creatine kinase isoforms by agarose gel electrophoresis in the diagnosis of myocardial infarction.

To evaluate a method to quantitate the isoforms of serum creatine kinase isoenzyme MM (CK-MM) by agarose gel electrophoresis, sera of normal subjects (n = 74) and patients with acute myocardial infarction (n = 21) and other diseases (n = 67) were studied. The within-assay imprecision (CV) for CK-MM1, -MM2, and -MM3 was 1.9%, 0.8%, and 2.2% at the activity of 79, 105, and 64 U/L (30 degrees C, CK-NAC), respectively; while the assay-to-assay imprecision was 4.8%, 3.2% and 3.9%, respectively. The method could detect 5 U/L or more of any CK-MM isoform and was linear with respect to CK activity at values less than 1100 U/L. Sera from healthy subjects (n = 74) contained mainly CK-MM1 (mean = 48.5%), with lesser amounts of CK-MM2 (mean = 30.6%) and CK-MM3 (mean = 20.8%). The central 95-percentile reference range for the ratio of MM3/MM1 was 0.12-1.34 with mean = 0.49. The sensitivity of CK-MM3/MM1 ratio greater than 1.3 in the diagnosis of acute myocardial infarction employing the first available sample was 90% at a specificity of 91%, compared with a sensitivity of 81% and specificity of 87% for the conventional CK-MB assay. At CK-MM3/MM1 ratio of 1.6 or more, the specificity increased to 96% while sensitivity remained unchanged at 90%. This procedure for the quantitation of serum CK-MM isoforms is convenient, practical and suitable for inclusion in the routine panel of cardiac tests.     

Diagnostic use of CK-MM and CK-MB isoforms for detecting myocardial infarction

Following acute myocardial infarction, total CK and CK-MB levels begin to rise 5 to 6 hours after the onset of chest pain. The serial profile of the rise and fall of both activities is nearly always indicative of AMI. The recent increase in the use of thrombolytic agents in an attempt to attain reperfusion of occluded coronary arteries alters the enzyme profiles observed in blood after AMI. After successful reperfusion a washout phenomenon occurs in which early restoration of blood flow to damaged myocardium causes an early rise in total CK and MB levels above the normal range 2 to 4 hours after AMI, with earlier and higher peak enzyme values. Recently reports have appeared describing numerous serum and plasma CK-MM and CK-MB isoform patterns after AMI. Following release from injured myocardium CK-MM3 and CK-MB2 (designated the tissue isoforms) are converted in the circulation to post-translation products (MM2, MM1, MB1, respectively). Studies have now shown that CK-MM isoform patterns provide a unique means of assessing the time of onset of necrosis and a monitor of the duration of enzyme release from the site of injury. Following AMI, MM3, the MM3/MM1 ratio, or both rises and peaks earlier than either total CK or CK-MB levels. During successful reperfusion, the rate of rise of CK-MM3 is more rapid and the MM3/MM1 ratio peaks earlier than without reperfusion. However, any concomitant release of CK-MM3 from skeletal muscle would decrease the clinical utility of MM isoforms in detecting myocardial damage. Recent advances in technology have shown that CK-MB2 rise parallels the CK-MM increase and also rises earlier than total CK and total MB levels and provides increased specificity for the myocardium. The full potential of the diagnostic utility of MM and MB isoforms will not be realized until a reliable, sensitive, simple, and rapid quantitative assay becomes available.     

Evaluation of a new automated system for the determination of ck-mb isoforms

We evaluated a new analyzer (Cardio REP) specifically designed for cardiac CK-MB isoenzyme and isoforms activity, with a performance time of 24 minutes. Ten AMI patients, with times elapsed between the onset of chest pain and admission to hospital ranging from 30 minutes to 4 hours, were monitored every 3–4 hours until the 16th hour of hospitalization. In each serum sample, in addition to total CK-MB and CK-MB isoforms measured by the Cardio REP analyzer, we also assayed total CK activity, CK-MB activity by immunoinhibition method, CK-MB mass concentration, CK-MB isoforms by REP method, troponin T, and myoglobin. The precision study demonstrated acceptable within assay and between assay CVs% for total CK-MB (8.1 and 10.4), MB₁ (9.1 and 14.2), and MB₂ (9.1 and 8.2) isoforms. The method was found to be linear up to 371 U/L for MB₂ isoform fraction and up to 516 U/L for total CK-MB. Results for CK-MB obtained with the Cardio REP correlated well with those for CK-MB activity obtained with the immunoinhibition method (r = 0.869) and those of CK-MB mass concentration (r = 0.923). The sensitivity of the Cardio REP CK isoforms method was found to be greater than that of the REP CK isoforms method. Time to first increased value of MB₂/MB₁ ratio and MB₂ isoform was earlier in comparison to that for CK-MB mass concentrations and similar to that for myoglobin, a marker that, however, lacks specificity. The diagnostic efficiency of CK-MB isoforms and the availability of a real-time, fully automated method for their measurement suggest the utilization of this biochemical marker in emergency for the early diagnosis of AMI.     

At what level of serum total creatine kinase activity can measurement of serum creatine kinase MB isoenzyme activity be omitted in suspected myocardial infarction?

The purpose of this study was to establish a discriminatory limit for serum total creatine kinase activity (CK activity) below which CK isoenzyme fractionation is unnecessary. We looked at 2610 serum samples from 1077 consecutive patients with suspected acute myocardial infraction (AMI). The CK activity was determined according to the Scandinavian recommended method. Isoenzymes of CK were separated by agarose gel electrophoresis, followed by fluorometric scanning. When the threshold for CK activity was 150 U/l, none of the samples had a creatine kinase MB isoenzyme activity (CK-MB activity) equal to or higher than 30 U/l (the diagnostic level), which has been found to differentiate between patients with AMI and those without AMI. Only 14 patients (1.3% of all patients investigated) had CK-MB activity peaks between 10 U/l (detection limit) and 30 U/l. Of these, AMI was only diagnosed in one. We recommend that CK-MB activity should be measured only when CK activity is higher than 150 U/l. This would make about 50% of all CK-MB measurements unnecessary.     

Monoclonal antibody inhibiting creatine kinase MM3 but not isoform MM1

Monoclonal antibody CKM-G01 inhibited greater than 99% of the activity of porcine and human creatine kinase(CK)-MM isoenzyme purified from muscle. However, it inhibited only 54% of CK-MM in human serum. Chromatofocusing of serum CK-MM showed that CKM-G01 inhibited 100% of MM3 but not isoform MM1. CKM-G01 inhibited CK-MM2 by 57%. CKM-G01 specifically inhibited only the original CK-M subunit and not the subunit modified by removal of C-terminal lysine by carboxypeptidase N. CKM-G01 can be used for assay of CK isoforms. We devised a new diagnostic reagent involving it, which requires no analytical separation of isoforms, based on the immunoinhibition method, and applied it to early diagnosis of acute myocardial infarction. The "inhibition index," (inhibited CK activity/total CK activity) x 100, increased more rapidly than did total CK and CK-MB. Evidently this diagnostic reagent can be used for easy, early diagnosis of acute myocardial infarction.     

肌酸激酶同工酶检测在急性心肌梗死中的临床价值

目的 探讨肌酸激酶（CK）MB和MM同工酶（CK-MB和CK-MM）亚型在急性心肌梗死（AMI）患者中的变化规律与其预后的关系,评价CK同工酶的亚型检测在AMI心肌早期再灌注、梗死延迟或再梗死诊断中的临床价值。方法 采用琼脂糖凝胶电泳系统将血清CK同工酶亚型分离为CK-MM3、CK-MM2、CK-MM1、CK-MB2和CK-MB1,并分析比较21例AMI患者血清CK同工酶亚型在发病后0～6小时、24小时和72小时的动态变化。结果 AMI患者血清CK-MB和CK-MM在发病后6h开始升高,其中以CK-MB2和CK-MM3升高为主,MB2／MB1〉1．36,MM3／MM1〉0．7;12-24小时达峰值,CK-MB／CK〉30％。15例早期再灌注的AMI患者血清CK、CK-MB和CK-MM在72小时下降至正常,但6例无早期再灌注患者仍处于较高水平,其中MB2／MB1〉1．29,MM3／MM1〉0．65。结论 CK同工酶的亚型检测能反映AMI患者心肌组织损伤的动态过程,可作为一项较灵敏的生化指标,有助于诊断AMI心肌早期再灌注、梗死延迟或再梗死。     

Creatine kinase MM isoforms in serum after cardiac surgery

We evaluated creatine kinase (CK; EC 2.7.3.2) MM3:MM1 isoform ratios in the serum of cardiac patients immediately after cardiac surgery for the diagnosis of perioperative myocardial injury. The mean ratio was 4.8 (range, 1.4-10.7) in 22 patients who had postoperative myocardial complications and 4.6 (1.3-9.6) in 66 patients who did not. By the first postoperative day the ratio had decreased substantially in both groups of patients. The isoform ratio did not correlate with the concentration of total CK, CK-MB, total lactate dehydrogenase (LD), or the incidence of LD1:LD2 or LD5:LD2 ratio reversal. Of these measurements, CK-MB and LD concentrations differed most between the groups of patients; parallel testing of CK-MB and LD showed an optimized sensitivity and specificity of 77% and 87%, respectively. We conclude that analysis for CK-MM isoforms does not add information in the period immediately after cardiac surgery; concentrations of CK-MB and LD correlate with myocardial injury, but the sensitivity and specificity of these measurements may not be high enough for clinical utility.     

Radioimmunoassay of creatine kinase isoenzymes in human serum: isoenzyme MM

Measurement of the mass concentration of serum enzymes by radioimmunoassay provides direct quantitation of specific isoenzymes and may be less subject to some of the limitations of traditional assay procedures for enzymes. We describe the development of a sensitive and specific radioimmunoassay for the muscle isoenzyme of creatine kinase, CM-MM, in human serum. CK-MM, purified from human skeletal muscle, was used to raise high-titer antisera and for iodination by the Chloramine T method. The radioimmunoassay required 50 microliter of sample, utilized a double-antibody separation method, and was completed in 24 h. Cross reactivity with CK-BB was virtually zero, 3--17% with CK-MB. The mass concentration of CK-MM in the serum of healthy subjects ranged from 36 to 1668 microgram/liter and correlated closely with total CK enzymatic activity. Serum concentrations of CK-MM from casually selected patients correlated less well with total CK enzymatic activity, suggesting the existence of other CK isoenzymes or the presence of inactive forms.     

Creatine kinase isoenzymes in human cerebrospinal fluid and brain

Extracts of normal brains obtained at autopsy and cerebrospinal fluid (CSF) from patients with global brain ischemia were analyzed for creatine kinase (CK; EC 2.7.3.2) isoenzymes. We used both qualitative and quantitative assays (electrophoresis and immunoinhibition). Brain extracts contained CK-BB isoenzyme and mitochondrial CK. In 54 CSF samples free of blood contamination and with total activities ranging from 7 to 2010 U/L (mean 202 U/L), virtually all of the CK activity was due to CK-BB, and none to CK-MM or CK-MB. We conclude that brain contains CK-BB and mitochondrial CK, but lacks CK-MM and CK-MB. After cardiac arrest, CK-BB is released into the CSF. Any CK-MM in the CSF is probably from blood contamination, in which case immunoinhibition with anti-CK-M antibodies accurately quantifies CK-BB.     

Analytical and clinical evaluation of creatine kinase MB mass assay by IMx: comparison with MB isoenzyme activity and serum myoglobin for early diagnosis of myocardial infarction.

We analytically and clinically evaluated Abbott's IMx assay for creatine kinase (CK) isoenzyme MB (CK-MB) in serum. Over a 1-year period, the method was more specific but less precise than catalytic isoenzyme measurements by electrophoresis or immunoinhibition. Sera from different individuals without electrophoretic evidence of CK-MB but containing macro CK type 1 (n = 20), mitochondrial CK (n = 5), or CK-BB (n = 5) were scored as CK-MB negative by the IMx. Likewise, CK-MB-negative by the sera remained so after addition of purified human CK-MM (< or = 7600 U/L) or CK-BB (< or = 8100 U/L). For 39 patients admitted for suspicion of uncomplicated acute myocardial infarction (precordial pain for < or = 4 h), the diagnostic performance of the IMx CK-MB assay on admission and 4 h later was superior to that of total CK activity and compared well with that of CK-MB activity measured by electrophoresis or immunoinhibition. An admission, myoglobin showed a higher diagnostic sensitivity, specificity, and predictive value than did CK-MB and was the most informative test. Diagnostic performance on admission and 4 h later was further improved by considering positivity for myoglobin and for CK-MB by IMx and for the change in each over the first 4 h of hospitalization as criteria. Twelve hours after admission, diagnostic performance was further improved for all CK and CK-MB methods but began to decline for myoglobin.     

Early detection of skeletal muscle injury by assay of creatine kinase MM isoforms in serum after acute exercise

We could detect skeletal muscle injury early after an acute exercise bout by measuring creatine kinase (CK, EC 2.7.3.2) MM isoforms in serum. Eleven men performed 120 alternating-arm, eccentric (muscle lengthening) biceps contractions with the intensity of each contraction being 110% of maximal concentric strength--a form of exercise previously shown to cause significant increases of CK in serum at 24 h and muscle soreness 48 h after exercise. Total CK and CK-MM isoform activities in serum were determined before and at 0.5, 0.75, 1, 1.5, 2, and 6 h after exercise. Using thin-film agarose gels and a rapid isoelectric focusing technique, we separated the MM isoforms into MM3 (skeletal muscle form), MM2, and MM1 (in vivo conversion forms). The isoforms reflected the MM form released into the serum from tissue as well as the conversion of one form to another. There were no significant increases in total CK from before to 6 h after exercise: 75 (SD 36) vs 91 (SD 33) U/L. However, CK MM3 in serum increased significantly (P less than 0.01) within 2 h after exercise from 22 (SD 6)% to 28 (SD 6)%. The MM3 to MM1 ratio also increased significantly (P less than 0.05) during this time, from 0.6 (SD 0.3) to 0.9 (SD 0.4). Thus, quantification of CK MM isoforms permitted very early detection of skeletal muscle enzyme release.     

Analyses of creatine kinase isoenzymes and isoforms in serum to detect reperfusion after acute myocardial infarction

Isoenzymes and isoforms of creatine kinase (CK, EC 2.7.3.2) were measured to assess reperfusion after acute myocardial infarction (AMI). In streptokinase-treated and in spontaneously reperfused AMI patients, total CK, CK-2 activity and concentration, and CK-3(3) isoform activity peaked significantly (p less than 0.05) earlier than conventionally treated, non-reperfused patients. The ratio for CK-3(3) to CK-3(1) activities peaked significantly (p less than 0.05) earlier in both the streptokinase-treated and spontaneously reperfused groups, and indicated a greater release of enzyme (higher ratio) than in the conventionally treated patients. The ratio of CK-3(3)/3(1) also peaked significantly (p less than 0.05) earlier in all three groups than did total CK, CK-2, and CK-3(3) activities or concentrations. The clearance rates of total CK, CK-2, and CK-3(3) were not significantly different in all three groups. Thus, the ratio CK-3(3)/3(1) was the earliest indicator of infarction in both reperfused and non-reperfused patients.     

AMI患者早期CK同工酶亚型的高压电泳图谱分析

目的探讨CK同工酶亚型在AMI胸痛发作后24h内的变化规律,为AMI患者的早期诊断提供依据。方法采用REP全自动高压电泳仪检测AMI胸痛发作后不同时间以及对照组的CK-MM1、CK-MM2、CK-MM3、CK-MB1、CK-MB2等指标并进行荧光扫描,同时在Olympus2700全自动生化分析仪上测定CK、CK-MB的总活性,对所得数据进行恰当的统计分析。结果在AMI患者胸痛发作24h内,CK同工酶亚型有一特殊的变化规律:4~6h内,CK-MB2、CK-MM3开始升高,8~12h达高峰,92%的病人CK-MB2/CK-MB1>1.5,同时91%的病人CK-MM3/CK-MM1>0.5。结论CK同工酶亚型CK-MM3/CK-MM1、CK-MB2/CK-MB1的检测可作为AMI早期诊断的指标。     

An immunochemical procedure for determination of creatine kinase 3(1) (serum-specific) isoform in human serum evaluated

Changes in the proportions of individual isoforms of creatine kinase (CK) in serum promptly reflect both myocardial infarction and coronary reperfusion. A new commercial kit has been introduced for measuring CK-3(1) isoform in serum (ISOFOR-MM, International Immunoassay Labs.). This is an immunochemical assay containing CK-3(1) specific monoclonal antibody, bound to magnetizable particles, used to immunoextract this isoform. The CK activity of the sample is measured before and after immunoextraction and the difference in the two values gives the measure of CK-3(1). Extraction of CK-3(1) was complete at less than or equal to 1200 U/L. Analysis of between-day imprecision gave CV between 2.9-7.9%. The method was not susceptible to interference by CK-3(2) and CK-3(3) isoforms, CK-2 isoenzyme, or mitochondrial CK. Reference interval for CK-3(1) (expressed as percent of total CK-3) was 42-69%. Correlation between percent CK-3(1) by isoform electrophoresis (x) and evaluated procedure (y) was y = 0.83x + 7.6, with r = 0.957 (n = 40). The ISOFOR-MM performed well enough in this evaluation to replace electrophoresis or isoelectric focusing for measurement of CK-3(1) isoform.     

Cathodic isozyme of serum creatine kinase in a case of stomach cancer complicated by disseminated intravascular coagulation

A rare isozyme of serum creatine kinase (CK) migrating cathodic to CK-MM on electrophoresis was found in a 30-year-old male with stomach cancer complicated by disseminated intravascular coagulation leading to massive upper gastrointestinal bleeding and marked anemia. Serum CK activity rose to a maximum of 374 U/l without detectable CK-MB isozyme. The patient was also characterized by a marked increase in serum lactate dehydrogenase (all isozymes elevated) and by preferential leakage of mitochondrial aspartate aminotransferase and glutamate dehydrogenase, indicating the presence of extensive tissue damage involving mitochondria. Skeletal muscle mitochondria were considered the most likely source of the additional CK isozyme.     

Isoforms of creatine kinase MM and MB in acute myocardial infarction: a clinical evaluation

Serum creatine kinase (CK, EC 2.7.3.2) isoenzymes MM and MB were resolved, respectively, into three (MM1, MM2, MM3) and two (MB1, MB2) isoforms (subforms derived from the same isoenzyme) by electrophoresis and the isoform patterns were determined in multiple sequential serum samples, timed from the onset of chest pain, from 58 patients with acute myocardial infarction (AMI). During the first 3 h after the onset of chest pain, the serum isoform activity resembled the pattern seen in normal volunteers. Specimens obtained 6 h after AMI showed predominantly MM3 and MB2 (45% and 11% of the total CK activity, respectively). Between 10 and 72 h, there was a gradual shift in which MM3, MM2 and MB2 decreased, while MM1 and MB1 increased. MB2 and MB1 disappeared from the pattern for samples collected after 24-48 h, while MM1 was always the most prominent band at the end of the observation period (66%, range 41-77%, at 48 h). These data suggest that a single determination of CK isoform pattern, drawn between 6 and 48 h after AMI, may provide an effective means of predicting the time of onset of necrosis. There were no significant differences in the CK isoform patterns according to infarct location and functional status of patients.     

Adenylate kinase mimics creatine kinase-MM isoenzyme in a CK isoenzyme electrophoresis assay

Adenylate kinase activity (AK) originating from erythrocytes, present in hemolyzed serum behaves like creatine kinase MM isoenzyme (CK-MM) in some CK electrophoresis assays that employ, in their visualization reagent kits, adenosine monophosphate (AMP) as the sole inhibitor of AK, rather than a combination of AMP and a more potent inhibitor of erythrocyte AK, diadenosine pentaphosphate (Ap5A), to inhibit all contaminating-AK activities in serum and quantify only the CK isoenzyme activities in serum following electrophoretic fractionation on agarose gel. This can spuriously overestimate the CK-MM fraction and thereby result in underestimation of CK-MM or CK-BB isoenzymes if present. A hemolyzed serum sample obtained from an elderly patient was erroneously reported as containing low CK-MB due to such overestimation of CK-MM fraction in the sample. Supplementing the AMP already present in the visualization reagent formulation, used to estimate CK isoenzyme concentration in serum, with Ap5A can eliminate or effectively minimize AK interference, especially that caused by hemolysis, and thereby prevent reporting false-negative CK-MB result obtained with CK isoenzyme electrophoresis assays. © 1994 Wiley-Liss, Inc.     

Preparation and stability of a liquid creatine kinase isoenzyme control from rabbit serum.

The MB isoenzyme of creatine kinase (CK) may be prepared in vitro from rabbit serum containing only the MM and BB isoenzymes, by means of a hybridization technique. The MM and BB dimers dissociate in 4 mol/L urea, which allows random recombination of M and B monomers. A liquid CK-isoenzyme control can be made from mixtures of rabbit sera obtained after hybridization and stabilized with glycerol and 25 mmol of 2-mercaptoethanol per liter. A liquid control stored at 4 degrees C showed good stability over a three-month period, declining to a mean residual activity of CK of approximately 90% after three weeks and a mean residual activity of MM, MB, and BB of 80--85% after six weeks. At 25 degrees C, CK activity of the liquid control declined to 75--80% after the fourth week. CK-BB at 25 degrees C was the least stable isoenzyme, declining to 75% after the third week and reaching 60% of activity after 12 weeks. CK-MB and CK-MM showed approximately 10--15% less stability at 25 degrees C than at 4 degrees C.     

An immunologic pathway for intravascular catabolism of creatine kinase subform MM3.

In vitro incubation of the MM3 subform of human creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2, CK) with fresh human serum resulted in the formation of a complex of high relative molecular mass (Mr 320 kDa). The formed complex (macro CK-MM3) consists of both CK-MM3 and immunoglobulin A (IgA), and its amount of the formed complex was proportional to CK-MM3 activity and IgA concentration. Two molecules of CK-MM3 combined with one molecule of IgA, and the immunoglobulin inhibited the enzyme activity. As IgA does not form complexes with other subforms (CK-MM2 and CK-MM1) or CK-MB, the antigen specificity of IgA to CK-MM3 is definitely exacting. The circumstantial evidence suggests that macro CK-MM3 is a specific antigen-antibody complex. Macro CK-MM3 was detected in all of the examined sera of adult patients with more than 2001 U/1 CK activity (the positive percentage of macro CK-MM3 in all adult patients was 73%), but not detected in sera of patients who were younger than 12 months old. No relationship was observed between macro CK-MM3 and the patients' underlying diseases. Macro CK-MM3 formation suggested to be an immunologic pathway for intravascular catabolism of CK-MM3 when its activity increases.