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7 d

Single value above 99th percentile of control reference population is surrogate of myocardial necrosis in setting of myocardial ischemia

a Time of first increase for the markers was 1-3 h for myoglobin, 3-4 h for CK-MB mass, 3-4 h for cTnT, and 4-6 h for cTnl. b +, modest cardiac specificity; ++, high cardiac specificity; +++, very high (absolute) cardiac specificity.

LD2 isoenzymes predominated in serum after MI, frequently in an LD1 > LD2 flipped pattern, whereas LD4 and LD5 predominated in patients with hepatic disease or skeletal muscle injury and LD2, LD3, and LD4 with involvement of platelets or lymphatic disease (11). Fractionation of LD, focusing on the LD1 and LD2 isoenzymes, was used as an aid for the diagnosis of MI into the 1980s.

Total activity of LD is a relatively late biomarker with concentrations generally increasing to above normal levels in 24-48 h after coronary occlusion, peaking in 3-6 d, and returning to normal in 8-14 d. The LD1 > LD2 flipped pattern is elevated earlier, at 1012 h after MI, peaking in 2 to 3 d, and returning to normal within 7-10 d (11,12). Using the MI diagnostic criteria that was standard in the 1980s, an elevated LD1 level and a flipped ratio had a sensitivity and specificity of approx 75-90% (11,12).

Measurement of LD activity and LD1/LD2 isoenzyme fractionation allowed a prolonged retrospective diagnosis of MI, which was useful for assessment several days after the acute event. However, the advent of reliable assays for cardiac troponin rendered LD measurement and fractionation for the diagnosis of MI an anachronism (13). According to the recent ESC/ACC redefinition of MI, total LD activity and LD isoenzymes should not be used for the diagnosis of cardiac damage (6).

Myosin Light Chains

The sarcomere is the basic building block of the contractile apparatus in both skeletal and cardiac muscle. Differences in contraction characteristics of cardiac and skeletal muscle led to the notion that there were different sacromeric isoforms for the protein myosin in these tissues (14). Myosin consists of two heavy chains and two pairs of light chains termed myosin light chain 1 (MLC1), with a molecular mass of 27 kDa, and MLC2, with a molecular mass of 20 kDa. The MLCs are structural proteins that modulate interaction between myosin and actin. Interest in MLC was stimulated by the development of antibodies that reportedly differentiated cardiac from skeletal muscle MLC (15). Although <1% of MLC is found in the cytosol, this compartment serves as a staging area for myosin synthesis. MLC from the cytosol is released into the circulation 3-6 h after acute necrosis and remains elevated for 10-14 d, as the structural component is gradually released after cell death; peak MLC concentrations occur after 4 d (16).

Cardiac MLC showed promise for risk stratification and as a biomarker of necrosis. Hillis et al. (17) measured cTnI, MLC1, and creatine kinase (CK)-MB mass at presentation and then 4, 8, 16, and 24 h later in 208 patients with chest pain without new ST-elevation. Both cTnI and MLC1 predicted the long-term outcome of patients with chest pain, and in this cohort MLC1 was a better predictor of mortality and nonfatal acute MI than the measurement of cTnI. Although MLC measurements were initially received enthusiastically by the cardiology and laboratory medicine communities (18,19), in the 1980s it was suspected and later verified that the apparent cardiac isoform of MLC is also produced by slow-twitch skeletal muscle, thus explaining the observed crossreactivity in some clinical samples (20). MLC measurements therefore fell out of favor compared with CK-MB and as assays for cardiac troponin were being developed.

Aspartate Aminotransferase

Aspartate aminotransferase (AST) was among the earliest myocardial cell necrosis biomarkers utilized in the clinical laboratory (Fig. 2) (21). AST (or SGOT) is a member ofthe aminotransferases; such enzymes catalyze the conversion of amino acids into 2-oxo-acids by transfer of amino groups. AST is abundant in skeletal muscle, liver tissue, erythrocytes, and myocardium (11). Distinct AST isoenzymes are present in the cytoplasm and mitochondria of cells; the half-life of the mitochondrial form is 10 d, vs the cytoplasmic form, which has a shorter half-life of only 10 h. Most experts agree that modest tissue injury causes release of the cytoplasmic isoenzyme, whereas more extensive damage results in release of the mitochondrial isoenzyme as well. The isoenzymes are not fractionated for clinical use.

AST is measured with activity assays. AST activity is normally present in serum, cerebrospinal fluid, and saliva but not in urine unless there is substantial renal injury. Heart tissue has high AST activity, but as stated, the enzyme is not specific for myocardial tissue. After MI, AST becomes abnormal in circulation within 6-8 h after the onset of symptoms, with peak values four- to fivefold higher than the upper limit of normal (ULN) reached after 18-24 h, and levels returning to normal within 4 to 5 d (12). Utilization of AST for diagnosis, monitoring, risk stratification, or other purposes in the context of MI is an anachronism because more sensitive and specific biomarkers have become available. According to the recent ESC/ACC redefinition of MI, AST should not be used as a biomarker to diagnose MI (6).

necrosis biomarkers of the present

CK: Total CK and the CK-MM, CK-MB, and CK-BB Isoenzymes

Total CK comprises three major isoenzymes that catalyze the following reaction:

Creatine + ATP CK, Mg++ ADP + Creatine Phosphate pH = 6.8

When cellular energy demands are relatively low and intracellular pH is approx 7.0, CK catalyzes the forward reaction with the formation of creatine phosphate. Creatine phosphate can be viewed as a storehouse for high-energy phosphate bonds that have been transferred from adenosine triphosphate (ATP). As indicated in the equation, CK also catalyzes the reverse reaction. When demands for energy in the cell increase, there is a rapid drop in intracellular pH caused by the local formation of acidic compounds, which induces CK to catalyze transfer ofa high-energy phosphate bond from creatine phosphate to adenosine 5'-monophosphate (ADP), thus producing intracellular ATP in order to meet the increased energy demands.

CK is abundant in tissues that have periods of relative quiescence and then bursts of brisk activity or in cells subjected to differences in oxygen gradient. Expressed in terms of grams of wet tissue, total CK activity is highest in skeletal muscle, at 2500 U/g; heart has 473 U/g and brain has 55 U/g (11). A variety of other tissues such as the small intestine, tongue, diaphragm, uterus, and prostate also contain total CK activity, but in lower relative amounts (11). CK activity is virtually absent in most other tissues. Total CK tissue-to-plasma ratio is very high in skeletal muscle and somewhat high (albeit sevenfold less) in myocardium, conferring good performance as a biomarker, because dramatically increased plasma values are observed in association with increased muscle necrosis and trauma. Serum total CK concentrations are dependent on muscle mass, and average levels are higher in men than in women, higher in African Americans than in Caucasians, and higher in younger adults than in the elderly (11). In a study of marathon runners, substantial increases in total CK levels were observed after extreme exercise, compared with prerace levels; concentrations returned to baseline in 4 d (22). There is no evidence that marathon running damages healthy, well-perfused myocardium, despite the fact that the plasma levels of total CK and other biomarkers may increase to the same as those measured after MI (23). However, these indices must be interpreted with caution for individuals who habitually undertake strenuous exercise, especially if they have done so within the previous week (23). Rhabdomyolysis also causes greatly increased serum total CK activity, as well as other skeletal muscle biomarkers such as myoglobin (11).

According to the ESC/ACC redefinition of MI, measurement of total CK is not recommended for the routine assessment of MI, because its wide tissue distribution confers lower diagnostic accuracy compared with other biomarkers (6). Some clinicians continue to measure total CK for epidemiological or scientific use, in which case the cutoff limit should be at least twice the ULN (6). Because the test is inexpensive and does not require specialized analyzers, as do other cardiac biomarkers, utilization of total CK for the diagnosis of MI may be reasonable in developing countries.

CK-MB Isoenzyme

Total CK activity represents the cumulative concentration of three CK isoenzymes. CK is a dimeric enzyme consisting of two types of subunits designated either "M," originally for "muscle," or "B" to connote "brain." Each of these subunits is produced by a distinct gene (11). The nature of CK gives rise to three major isoenzymes: the two homod-imers CK-MM and CK-BB and the heterodimer CK-MB (9). In patients with significant myocardial disease, i.e., aortic stenosis, coronary artery disease, or both, the CK-MB isoenzyme comprises approx 20% of the total CK in this tissue (24), whereas CK-MB comprises <3% of CK in skeletal muscle (25). Although healthy individuals may have a much lower percentage of CK-MB of1.1% in their heart tissue (24), the far higher and consistently elevated CK-MB fraction in vulnerable patients with significant coronary heart disease confers excellent myocardial tissue specificity. CK-BB is found primarily in brain and intestinal tissue and contributes little to total CK activity; CK-BB is virtually absent from skeletal or cardiac tissue (25).

In skeletal muscle, usually >99% ofthe CK activity comprises the CK-MM isoenzyme (25). Although myocardium is the only tissue that has both a high proportion and high concentration of CK-MB, injury to skeletal muscle can compromise the diagnostic use of this biomarker because of the sevenfold higher total CK activity on a per-gram basis and the potential for the release of substantial CK-MB upon injury. Further, the body mass of skeletal muscle tissue is approx 100-fold greater than of myocardial muscle. To provide greater cardiac specificity using CK-MB, a CK-MB index may be calculated according to the following equation:

CK-MB index values exceeding 2.5% are usually associated with a myocardial source of the MB isoenzyme (26); however, the index is reportedly as low as 2% and as high as 5%, depending on variability of both the numerator and denominator (27). A particular problem is posed by patients with both myocardial and skeletal muscle injury because CK-MB release from skeletal muscle may confound the diagnostic use of the CK-MB index by masking the relatively subtle CK-MB contribution from heart tissue and effectively "swamping" the denominator. It is also of note that elevations in both CK-MB concentration and CK-MB index have been attributed to skeletal muscle injury alone, with no

Fig. 3. (A) CK-MM isoforms. CK-MM3 consists of two unmodified tissue M subunits. On release into the circulation, carboxypeptidase N (CPN) hydrolyzes a terminal lysine at the C-terminus of each M subunit to generate the modified serum forms: MM2 (missing one lysine) and MM1 (missing two lysine residues, one from each polypeptide chain). Plus and minus signs represent the presence and absence of lysine residues, respectively. (B) CK-MB isoforms. Carboxypeptidase N (CPN) cleaves a lysine from the C-terminus of the M subunit of the MB2 (tissue isoform) and generates the MB1 (serum isoform). (C) Immunoinhibition of CK-MB activity. Anti-CK-M antibodies selectively bind to and inhibit the enzymatic activity of the CK-M subunits. The residual CK activity, measured by using total CK reagents, is the result of the B subunits of MB alone. To calculate the activity of the intact CK-MB isoenzyme, the residual result must be multiplied by a factor of two. (D) CK-MB mass immunoassay. Anti-CK-MB is immobilized on a solid matrix. CK-MB in the sample binds to the solid-phase antibody. Anti-CK-MM conjugate is added and binds to the antibody-CK-MB complex to form an antibody-CK-MB-antibody conjugate complex. The conjugate enzyme acts on the substrate to produce a colored product or fluorescence.

Fig. 3. (A) CK-MM isoforms. CK-MM3 consists of two unmodified tissue M subunits. On release into the circulation, carboxypeptidase N (CPN) hydrolyzes a terminal lysine at the C-terminus of each M subunit to generate the modified serum forms: MM2 (missing one lysine) and MM1 (missing two lysine residues, one from each polypeptide chain). Plus and minus signs represent the presence and absence of lysine residues, respectively. (B) CK-MB isoforms. Carboxypeptidase N (CPN) cleaves a lysine from the C-terminus of the M subunit of the MB2 (tissue isoform) and generates the MB1 (serum isoform). (C) Immunoinhibition of CK-MB activity. Anti-CK-M antibodies selectively bind to and inhibit the enzymatic activity of the CK-M subunits. The residual CK activity, measured by using total CK reagents, is the result of the B subunits of MB alone. To calculate the activity of the intact CK-MB isoenzyme, the residual result must be multiplied by a factor of two. (D) CK-MB mass immunoassay. Anti-CK-MB is immobilized on a solid matrix. CK-MB in the sample binds to the solid-phase antibody. Anti-CK-MM conjugate is added and binds to the antibody-CK-MB complex to form an antibody-CK-MB-antibody conjugate complex. The conjugate enzyme acts on the substrate to produce a colored product or fluorescence.

evidence of myocardial involvement (11). This pattern is infrequent, however, and overall CK-MB has been an excellent biomarker of myocardial injury over several decades.

CK-MB Activity and Mass Assays

CK-MB measurements are considered highly selective for myocardium and in the 1970s became the standard for diagnostic assessment of MI (Fig. 2) (20). M subunits have a different charge relative to B subunits, and this difference in charge was used to fractionate total CK into serum CK-MM, CK-MB, and CK-BB isoenzymes using the principles of electrophoresis and ion-exchange column chromatography (28). Strategies based on measuring CK activity after separating out CK-MB were developed and used extensively from the late 1970s through the mid-1980s, when the first immunobased assays appeared in which antibodies were used to assess CK-MB activity. CK-MB activity assays used antibody reagents to inhibit activity of the M subunits of CK-MM and CK-MB (Fig. 3). Subsequently, total CK activity was measured, and assuming that CK-BB was negligible, all the activity remaining in the sample was attributed to the B subunit of CK-MB.

Results were multiplied by 2 to compensate for the activity of the inhibited M activity. These activity assays led to incorporation of CK-MB into updated criteria for the diagnosis of MI (29).

Although a very important contribution, CK-MB activity assays were and are limited by their inability to detect CK-MB at low concentrations. In addition, electrophoresis and column chromatography methods were subject to false-positive interference from so-called macro-CK, predominately caused by an immune response against CK-BB in some populations (11). Immunoinhibition assays were subject to interference from the presence of macro-CK as well as from any CK-BB in patient samples. For these reasons, CK-MB activity assays gave way to more analytically sensitive and specific mass immunoassays that offered the advantage ofquantifying CK-MB as a specific protein, rather than relying on functional enzymatic activity as a surrogate. CK-MB activity assays have been replaced by CK-MB mass assays and by cardiac troponin; however, they are relatively inexpensive and, therefore, remain in use in some areas owing to economic conditions (6).

The earliest mass assay was a radioimmunoassay (RIA) that targeted the B subunit of CK-MB (30). This assay set the stage for the development of CK-MB-specific antibodies, the most widely used of which is the Conan antibody that is a component of many commercial assays in use today (31). Most mass assays are based on incorporation oftwo antibodies, one that captures the CK-MB in patient samples and another to which a signaling reagent is bound for detection (Fig. 3). In this way, a capture Ab-CK-MB-signal Ab sandwich is formed and detected. A standard dose-response curve is plotted using CK-MB standards from which the analytical concentration can be related to the mass of the CK-MB protein.

CK-MB Isoforms

Myocardial tissue contains one CK-MB protein isoform. After release into the circulation after cell death, the M subunit of CK-MB from tissue undergoes posttranslational modification through cleavage of the C-terminal lysine by the blood enzyme carboxy-peptidase-N (32). This modification forms a differently charged form of CK-MB, termed CK-MB 1, that can be separated from the tissue form, termed CK-MB2, by electrophoresis (32). Studies characterizing the clinical performance of the isoforms of CK-MB isoenzymes (CK-MB1 and CK-MB2) show promise for earlier identification of myocardial damage than assays of CK-MB alone. In homeostatic conditions, serum CK-MB2/CK-MB1 ratios are approx 1.0, and total CK-MB concentration is <1.5 IU/L (33). Release of the CK-MB2 tissue isoform into the circulation from damaged myocardial tissue increases the absolute level of CK-MB in the blood and also increases the ratio of CK-MB2/CK-MB1. The increased CK-MB2/CK-MB1 ratio may be detected as early as 1 to 1.5 h after the onset of chest pain (33). The change in isoform ratio may precede significant elevations in CK-MB by up to 4 h, providing earlier identification of MI. Using a 2.6 IU/L CK-MB cutoff with a CK-MB2/CK-MB1 ratio © 1.5, a study of1100 patients presenting with signs and symptoms of ACS showed values of 6-h sensitivity and specificity for diagnosing MI of 95.7 and 93.9%, respectively (34). Respective sensitivity and specificity for conventional 6-h CK-MB were reportedly 48.2 and 94% in this same population (34).

Automated processing for CK-MB isoforms is available, but interpretation can be technically challenging and the assay is available in only a few institutions. Both the National Academy of Clinical Biochemistry (35) and the ESC/ACC (6) have suggested consideration of myoglobin or CK-MB isoforms for use in the early diagnosis of myocardial injury.

CK-MB Mass for Diagnosis of MI

Figure 4 displays the biomarker release profile after MI. Release of CK-MB into the circulation occurs with the death of myocardial cells but does not occur with myocardial ischemia (36). In the past, CK-MB mass assays were considered the "gold standard" for the diagnosis of MI until reliable cardiac troponin assays became proven and widely available (37). The first rise in CK-MB following MI occurs 4-6 h after the onset of symptoms, but serial sampling over a period of 8-12 h is required for high sensitivity (6). Thus, despite excellent clinical performance, CK-MB is not an early marker and tissue specificity remains an issue in some cases. These assays are automated, have excellent detection limits (<1 ^g/L), are specific for CK-MB, and provide a rapid turnaround time (as short as 7 min). The American Association for Clinical Chemistry has proposed a recombinant CK-MB material for standardization of CK-MB mass assays (38).

Measurement of CK-MB mass remains a sound tool for the evaluation of MI (6), but most healthy individuals have a measurable amount of CK-MB biological "background noise" in their blood, probably from skeletal muscle turnover. For diagnostic use, CK-MB release from myocardium (i.e., signal) must substantially exceed this noise, which renders CK-MB less diagnostically sensitive compared with cTnT or cTnI, for which the physiological background noise is virtually zero. The signal/noise difference between CK-MB and cardiac troponin is illustrated in Table 3, which shows an analysis of six cohorts that directly compares these biomarkers (39). In nearly 18,500 patients, measurement of cardiac troponin detected more MIs than did CK-MB in all six cohorts (range: +12 to+127%), with an overall increase of 14% more MIs diagnosed using cardiac troponin.

A meta-analysis focusing on the performance of CK-MB mass measurements in populations from emergency departments or inpatient cardiology units was conducted in 1995 and showed a diagnostic sensitivity of 96.8% (95% confidence interval [CI]: 95-98%) and a diagnostic specificity of 89.6% (95% CI: 87-92%) (40). However, this diagnostic performance is no longer accurate, because over the past decade it has become clear that even small amounts of necrosis, and the associated small release of cTnT and cTnI, identify patients at high risk of adverse events (41,42). Thus, cTnT and cTnI are the preferred markers, and CK-MB has been replaced as the "gold standard" for the diagnosis of MI (Table 2) (6). Nevertheless, many clinicians believe that measurements of CK-MB mass

Table 3

Portion of Positive Cardiac Troponin and CK-MB Results for Patients With Suspected MI From Five Databasesa

Table 3

Portion of Positive Cardiac Troponin and CK-MB Results for Patients With Suspected MI From Five Databasesa

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