Cardiac Biomarkers

Cardiac biomarkers–the old and the new:a review Vikas Singh,Pedro Martinezclark,Mario Pascual,Eric Scot Shaw and William W.O’NeillBiomarkers are biological parameters that can be objectively measured and quantified as indicators of normal biologic processes,pathogenic processes,or responses to a therapeutic intervention.Typically thought of as disease process screening,diagnosing,or monitoring tools,biomarkers may also be used to determine disease susceptibility and eligibility for specific therapies.Cardiac biomarkers are protein components of cell structures that are released into circulation when myocardial injury occurs. They play a pivotal role in the diagnosis,risk stratification, and treatment of patients with chest pain and suspected acute coronary syndrome and those with acute exacerbations of heart failure.Cardiac markers are central to the new definition of acute myocardial infarction put forward by the American College of Cardiology and the European Society of Cardiology.Active investigation has brought forward an increasingly large number of novel candidate markers but few have withstood the test of time and become integrated into contemporary clinical care because of their readily apparent diagnostic,prognostic,or therapeutic utility.Coron Artery Dis21:244–256 c2010 Wolters Kluwer Health|Lippincott Williams&Wilkins.Coronary Artery Disease2010,21:244–256Keywords:acute,coronary,novel,protein,syndromeUniversity of Miami,Miller School of Medicine,Miami,Florida,USA Correspondence to Dr Pedro Martinezclark,MD,University of Miami,Miller School of Medicine,1400NW12th Avenue,Suite1179,Miami,FL,33136,USA Tel:+13052435050;fax:+13052435578;e-mail:pmclark@Received4January2010Revised31January2010Accepted2February2010IntroductionBiomarkers are biological parameters that can be objec-tively measured and quantified as indicators of normal biologic processes,pathogenic processes,or responses to a therapeutic intervention.T ypically thought of as disease process screening,diagnosing,or monitoring tools,biomar-kers may also be used to determine disease susceptibility and eligibility for specific therapies.Cardiac biomarkers are protein components of cell structures that are released into circulation when myocardial injury occurs.They play a pivotal role in the diagnosis,risk stratification,and treatment of patients with chest pain and suspected acute coronary syndrome (ACS)as well as those with acute exacerbations of heart failure.Cardiac markers are central to the new definition of acute myocardial infarction(AMI)put forward by the American College of Cardiology and the European Society of Cardiology[1,2].Active investigation has brought forward an increasingly large number of novel candidate markers but few have withstood the test of time and become integrated into contemporary clinical care because of their readily apparent diagnostic,prognostic,and/or therapeutic utility (T able1).Markers for inflammation and plaque destabilizationC-reactive proteinC-reactive protein(CRP)is a protein found in serum or plasma at elevated levels during an inflammatory process (Fig.1).It is a sensitive marker of acute and chronic inflammation and infection and,in such cases,is increased several hundred-fold.Extensive studies beginning in the early1990s showed elevated CRP levels independently predicted adverse cardiac events at both the primary and secondary prevention levels.More than15prospectively conducted clinical studies have shown CRP to be associated with short-term and long-term morality risk not only for patients with acute and chronic ischemic heart disease but also for those at risk for atherosclerosis[3].Increases in CRP levels detected by assays with expanded sensitivity to very low levels of CRP,so-called high-sensitivity CRP(hs-CRP),showed a strong correla-tion as an independent risk factor for future cardiac events.New rapid tests for CRP at the ultrasensitive level have also been developed.Guidelines published by the Centers for Disease Control/AHA indicate that based on results using standardized assays with precision to or below0.3mg/l,cut points of low risk(<1.0mg/l),average risk(1–3mg/l),and high risk(>3.0mg/l)be assigned to those patients with an intermediate10-year CHD risk(10–20%Framingham Risk Score/Adult T reatment Panel III guidelines)[4].hs-CRP predicts new coronary events in patients with ACS and unstable angina(UA),AMI,and risk of res-tenosis after revascularization procedures,independent of troponin T[5].The estimations that more than30% of patients with severe UA do not present with elevated hs-CRP levels along with its nonspecific nature pose a limitation to its use[6].244Perspective0954-6928 c2010Wolters Kluwer Health|Lippincott Williams&Wilkins DOI:10.1097/MCA.0b013e328338cd1fMyeloperoxidaseReleased from activated neutrophils,myeloperoxidase (MPO)is a leukocyte enzyme possessing powerful pro-oxidative and pro-inflammatory properties that play important roles in the pathogenesis of destabilization of coronary artery disease(CAD).MPO catalyzes the conversion of chloride and hydrogen peroxide to hypo-chlorite.It has been implicated in the oxidation of lipids contained within LDL cholesterol and consumption of endothelial-derived nitrous oxide,thereby reducing nitrous oxide bioavailability and impairing its vasodilating and anti-inflammatory properties.The blood and leuko-cyte MPO activity is found to be higher in patients with CAD than angiographically verified normal indivi-duals[7].A recent study has shown an association of MPO levels with the risk of future CAD in an apparently healthy population[8].Thus,even in the absence of myocardial necrosis and in negative cardiac troponin patients,baseline measurements of MPO significantly enhance the identification of patients at risk.New rapid tests for MPO levels have been developed and studies suggest that a value of more than350m g/l is associated with a considerably increased risk of heart attack[9].MPO plays a role in the degradation of the fibrous cap,making it both a marker of inflammation (neutrophil activation)and plaque instability(that precedes ACS).This property makes MPO a useful marker for short-term risk stratification.F urther validation studies on MPO in the general emergency department (ED)chest pain population are needed to determine its sensitivity,specificity,positive predictive value,and negative predictive value(NPV).Matrix metalloproteinasesMatrix metalloproteinases(MMPs)are a family of proteolytic enzymes that cleave the extracellular matrix and have been shown to be regulated by a class of proteins called tissue inhibitors of metalloproteinases. The MMPs are found in most tissues and are regulated byTable1Clinical utility-based classification of cardiac biomarkersInflammation and plaquedestabilization Ischemia Early necrosis Intermediate/late necrosis Heart failureC-reactive protein(CRP) hs-CRP Ischemia-modified albumin Myoglobin CK-MB Brain natriuretic peptide(BNP)Myeloperoxidase Glycogen phosphorylase enzymeBB(GPBB)Creatinine kinase MBisoforms-CKMB2Cardiac troponins(cTn)cTnTcTnITerminal fragment ofprohormone of BNPMatrix metalloproteinases Free fatty acids Soluble CD-40L(sCD40L)Fatty acid binding proteinsPregnancy-associated plasma protein A Phospholipase enzymes(A–D) Lipoprotein associatedphospholipase A2Placenta growth factor Interleukin-6Fig.1Platelets: sCD40L Lipid core:PAPP-AIL-6Macrophage:MMPOMMP-22IMAGPBBFABPLP-PLA2Biomarkers for plaque destabilization and ischemia.FABP,fatty acid binding protein;FFA,free fatty acids;GPBB,glycogen phosphorylase enzyme BB in brain;IL-6,interleukin-6;IMA,ischemia-modified albumin;LP-PLA2,lipoprotein-associated phospholipase A2;MMP,matrix metalloproteinase; PAPP-A,pregnancy-associated plasma protein A;sCD40L,soluble CD40ligand.Cardiac biomarkers–the old and the new Singh et al.245transcription in response to growth factors,cytokines, and hormones,and,extracellularly,in the form of pro-hormones,whose breakdown occurs mostly in response to plasmin.They are sub-grouped based on substrate specificity and structure,with MMP-2and MMP-9, (gelatinases)being of most current interest in inflamma-tion and cardiac disease.Several studies have shown that extracellular matrix degradation by MMPs,specifically MMP-9,are involved in the pathogenesis of a wide spectrum of cardiovascular disorders,including athero-sclerosis,restenosis,cardiomyopathy,congestive heart failure,MI,and aortic aneurysm[10,11].Similar to MPOs,the proteinases MMP-2and MMP-9are released from macrophages within the atherosclerotic plaques and have attracted attention as markers of plaque rupture.In patients with ACS MMP-2and MMP-9,levels at hospital admission are found to be two to three-fold higher as compared to that of patients with stable angina pectoris [12,13].Studies measuring MMP-9have indicated it to be another risk factor for assessing the severity of CAD in patients with ST-elevation MI[14].Soluble CD40ligandThe CD40and CD40ligand(CD40L)system is expressed on a variety of cell types including activated platelets,vascular endothelial cells,vascular smooth muscle cells,monocytes,and macrophages.After ex-pression on the cell surface,CD40L is partly cleaved by proteases and subsequently released into the circulation as soluble CD40L(sCD40L)that can be detected in serum and plasma.The main source of circulating sCD40L is platelets[15].It also shows that the anti-platelet treatment using the glycoprotein IIb/IIIa re-ceptor antagonist abciximab is beneficial to patients with elevated sCD40L levels[16].Several clinical studies have consistently reported that sCD40L is elevated in patients with ACS and that it provides prognostic information with therapeutic implications independent of established cardiac markers,for example cardiac troponins[16].Furthermore,patients with UA have higher plasma concentrations of sCD40L than healthy volunteers or those with stable angina,and elevation of sCD40L in this setting indicates a higher risk for recurrent events[16,17].The current standard for recurrent MI prediction by simultaneous assessment of sCD40L and cardiac troponin I(cTnI)yields indepen-dent and complementary prognostic information,thus enabling more powerful prediction of adverse cardiac outcomes[18].Increases in sCD40L have also been shown in a number of different inflammatory processes, so it is not specific for cardiac inflammation.No commercially available kit has been approved by the FDA to date and more studies using large cohorts will be required to validate the clinical use of sCD40L indepen-dently or in combination with other markers in the prediction of cardiovascular events after ACS.Pregnancy-associated plasma protein A and placenta growth factorHuman pregnancy-associated plasma protein-A(PAPP-A) is a200kDa metalloproteinase belonging to the metzin-cin superfamily of zinc peptidases originally identi-fied in the serum of pregnant women before delivery [19–21].The role of PAPP-A in tissue other than placenta including fibroblasts,vascular smooth muscle cells, and male and female reproductive tissues has been explored[22,23].PAPP-A,found equally in men and women,is histologically abundant in eroded and ruptured plaques but is not expressed in stable plaques[24]. Pregnancy-associated protein A is one of six different proteases that degrades insulin-like growth factor binding proteins.This proteolytic degradation of the insulin-like growth factor binding proteins is considered the predominant mechanism for the release of bioactive insulin growth factor-1(IGF-1)[25].A large study has illustrated that decreases in IGF-1appear to be cardio protective,yet some research shows that increases in PAPP-A,which should also increase the bioavailability of IGF-1,may be a relevant marker for the presence and extent of coronary atherosclerosis[26].It is believed that PAPP-A is released during plaque destabilization and appears to be a valuable indicator of UA and AMI in patients lacking other indicators of necrosis[24].In a study of patients with angiographi-cally confirmed ACS,an elevated level of serum PAPP-A was a strong independent predictor of nonfatal AMI or death.Elevated PAPP-A levels were able to identify patients at risk[27].PAPP-A as a marker can detect plaque rupture before markers that indicate onset of MI and myocardial necrosis.This capability for early determination of event risk makes PAPP-A a promising novel cardiac biomarker with potential applications for CAD risk assessment,diagnosis,and management. Placental growth factor is a member of the vascular endothelial growth factor family that stimulates vascular smooth muscle cell growth,recruits macrophages into atherosclerotic lesions,upregulates production of tumor necrosis factor-a and monocyte chemotactic protein1by macrophages,and stimulates pathological angiogenesis [28].It appears to be an initiator of the inflammatory process and a promising biomarker of plaque formation and plaque rupture.In one study,elevated placenta growth factor(P1GF)levels not only identified patients with acute chest pain who developed ACS,but also those patients with an increased risk of recurrent instability after hospital discharge[29].In another study comprising over32000women,an elevated level of PlGF was associated with an elevated risk for cardiovascular disease and subsequent cardiovascular events over a14-year follow-up period[30].The definite value of PAPP-A and PlGF still needs to be proven in larger studies.246Coronary Artery Disease2010,Vol21No4Interleukin-6Interleukin-6(IL-6)is a cytokine,a nonantibody protein, and intercellular mediator.Cytokine IL-6is produced by a variety of cells in the body;plasma concentrations reflect both the intensity of plaque vulnerability to rupture and,following percutaneous coronary interven-tion,restenosis.Cytokine IL-6is involved in the patho-genesis of ACS and has the following effects:stimulating the linear production of fibrinogen and CRP,stimulating the macrophage to produce tissue factor and MMPs, platelet aggregation,adhesion molecules,tumor necrosis factor,and vascular smooth muscle cell proliferation[31]. Cytokine IL-6predicts future MIs in healthy men and total mortality in the elderly[32].Elevation of circulating IL-6is a strong and independent marker of increased mortality in acute coronary events[33,34].Current value of inflammation markers Aforementioned markers for inflammation and plaque destabilization are nonspecific to cardiac disease but have,time and again,proved to be useful adjuncts as diagnostic markers for ACS in the ED when used in combination with TnI and brain natriuretic peptide (BNP).hs-CRP is the most valuable among these and predicts new coronary events in such cardiac patients independent of troponin T.Elevated PAPP-A levels identify patients with UA even in the absence of elevations in cTn or hs-CRP levels;there are,unfortu-nately,no currently available tests in a point-of-care testing(POCT)format.Although increased MPO has a role superior to that of PAPP-A,CD40L,and cytokines,is still inferior to CRP[35].Markers for ischemiaThe aim of diagnostic markers is to identify patients with ACS even in the absence of myocyte necrosis evidence (Fig.1).Ischemia-modified albuminAlbumin loses its ability to bind transitional metals like copper,cobalt,nickel in its N terminus region as it undergoes conformational change because of ischemia. This alteration is most likely caused by hypoxia,acidosis, free radical injury or energy-dependent membrane dis-ruption[36–38].This decrease in binding capacity can be measured by addition of a specified amount of cobalt to the patient’s serum followed by a colorimetric assay that determines the amount of unbound cobalt.This assay has been reported to be positive within minutes of ischemia, peaking within6h,and remains elevated for up to12h, thus allowing detection before the development of myo-cardial necrosis[as evidenced by normal levels of creati-nine kinase isoenzyme(CK-MB),troponin and,myoglobin] [39–42].Studies have also shown that ischemia-modified albumin (IMA)is a sensitive biomarker for the identification of ACS in patients presenting to the ED with typical chest pain at rest[43].The albumin cobalt binding test has been approved by the FDA for use as a rule-out marker for acute myocardial ischemia.The optimum cut-off for IMA,for ruling out ACS,is85kU/l and the higher values of100kU/l or more can be used for risk stratification.A meta-analysis of current data has shown that the finding of a negative IMA result,negative cTnT measurements, and a normal or nondiagnostic ECG,has a high NPV for excluding ACS in the ED[44].It is estimated that approximately1–2%of the total albumin concentration in the normal population is IMA compared to6–8%in patients experiencing ischemia and it is also found to be elevated in most patients with cirrhosis,bacterial and viral infections,advanced cancers, stroke(brain ischemia),and end-stage renal disease. Studies on the use of IMA in patients with chest pain in the ED have found sensitivities that ranged from71to 98%,and specificities of45–65%,with a NPV of90–97% for ACS.Thus,the main limitation of IMA at the present time is its low specificity.Glycogen phosphorylase isoenzyme BBGlycogen phosphorylase(GP)plays an essential role in the regulation of carbohydrate metabolism by mobi-lization of glycogen[45].It catalyses the first step in glycogenolysis in which glycogen is converted to glucose-1-phosphate,utilizing inorganic phosphate.The physio-logical form of GP is a dimer,which is composed of two identical subunits.Three different GP isoenzymes have been described in human tissues:GPLL(liver),GPMM (muscle),and GPBB(brain).Although isoenzymes BB and MM are found in the heart,GPBB is predominant. However,tissue concentrations of GPBB in the heart and brain are comparable raising issues with specificity[46]. In cardiomyocytes,GP is associated with glycogen and the sarcoplasmatic reticulum and forms a macromolecular complex,the sarcoplasmatic reticulum glycogenolysis complex[47,48].The metabolic state of the myocardium determines the degree of association of GP with this complex.With the onset of tissue hypoxia,GP is thereby converted from a particulate,structurally bound form into a soluble,cytoplasmatic form[47,49].A high GPBB concentration gradient is immediately formed in the perisarcoplasmatic reticulum compartment and GPBB is released from cardiomyocytes upon an increase in the cell membrane permeability.This makes GPBB an early marker for detection of ischemic myocardial damage. GPBB is not a heart-specific marker.However,its increase is rather specific for ischemic myocardial injury when damage to the brain and consequent disturbance of the blood–brain barrier can be excluded.GPBB levels increase between1and4h from chest pain onset and return to the reference interval within1–2days after AMI onset[50,51].Similar to other soluble markers, Cardiac biomarkers–the old and the new Singh et al.247such as myoglobin and CK-MB,it can be shown that GPBB time courses in AMI patients are markedly influenced by the fact of whether early reperfusion of the infarct-related coronary artery occurs.The acceler-ated GPBB release from cardiomyocytes after successful thrombolysis leads to a more rapid increase in GPBB, earlier and usually also higher peak values.GPBB thus may be useful,along with other soluble myocardial proteins,for assessing the effectiveness of thrombolytic therapy noninvasively.GPBB also increases,early on,in patients with UA and reversible ST-T alterations in the resting ECG at hospital admission and could be useful for early risk stratification in these patients[51,52]. GPBB is a promising marker for the early diagnosis of ACSs and could probably act as a marker of ischemia. However,further studies on specificity and development of a fast,automated assay are necessary before GPBB can be recommended as a routine diagnostic tool[53].Free fatty acidsFree fatty acids(FFAs)play several essential roles in physiologic homeostasis.Plasma long-chain fatty acids are either esterified to glycerol or nonesterified(or FFAs), most of which are bound to albumin.Under aerobic conditions,nonesterified long-chain FFAs represent the primary metabolic sources for the myocardium,account-ing for almost two-thirds of the ATP generated[54].The mechanism for uptake of FFAs into myocytes remains unclear but involves passive diffusion and/or active carrier-mediated transport.In the cytoplasm,long-chain FFAs are bound to fatty acid binding proteins(FABPs), which presumably facilitates their transport to the outer mitochondrial membrane where they become esterified/activated by long-chain acyl-CoA synthetase. Once activated,acyl-CoA esters are directed mainly to b-oxidation,but some may be stored as triglycerides or converted into membrane phospholipids.During hypoxia and ischemia,nonesterified fatty acids/FFAs have dama-ging effects on heart tissue and have been associated with an increased incidence of ventricular dysrhythmias and death in patients with AMI[55,56].Although most of the FFAs in serum are bound to albumin,a small amount is unbound;this is frequently referred to as the‘free’fraction.Serum FFAu concentrations are determined from the ratio of total serum FFAs to total serum albumin [57].Several studies suggest that increased unbound FFA concentration(FFAu),which is determined from the ratio of total serum FFAs to total serum albumin,may provide an early indicator of myocardial ischemia[58].Although the exact mechanism is not clear,it has been suggested that increased unbound FFAs result from catecholamine stimulated lipolysis in conjunction with a reduction of FFA utilization in myocardial ischemia.A POCT method for the determination of unbound FFA has already been developed(FFA Sciences)and has turned out to be quick (turnaround-time<1min)and precise(coefficient of variation7%)while requiring as little as15m l of plasma [59,60].This method uses a recombinant fatty acid binding protein labeled with a fluorescent probe of FFAu termed acrylodated intestinal fatty acid binding protein.When FFA binds to acrylodated intestinal fatty acid–binding protein,the fluorescent tag is displaced, thus producing a spectral shift that can be measured with a hand-held reader.Current data,although limited,suggest that monitoring of FFAu concentrations in patients presenting with ischemic symptoms may provide an early indication of cardiac ischemia.Fatty acid binding proteinsFABPs are relatively small(15kDa)intracellular proteins that are abundantly produced in tissues having active fatty acid metabolism,including the heart,liver,and intestine[61].FABPs bind long-chain fatty acids rever-sibly and noncovalently.Currently,nine distinct FABP types have been identified,with each type showing a characteristic pattern of tissue distribution and a stable intracellular half-life of2–3days[61].Several studies investigated H-FABP’s application as a biochemical marker of myocardial injury after it was first shown to be released from injured myocardium in1988.The H-FABP isoform is produced not only in cardiomyocytes but also,to a lesser extent,in skeletal muscle,distal tubular cells of the kidney,specific parts of the brain,lactating mammary glands,and placenta[62–64].H-FABP is not found in the circulation(plasma concentration<5m g/l)under nonpathological conditions and is rapidly released after AMI.H-FABP secretion into the interstitial space may be mediated by increased permeability of the myocardial cell membrane associated with severe ischemia[65].It has been shown that sequential H-FABP monitoring by only two samples,at admission and1h after admission, can reliably diagnose AMI within1h with a0.995area under the curve value in receiver operating characteristic curve analysis.In addition,100%of non-AMI can be excluded with no false-negative results[66].Therefore, H-FABP has been touted as an alternative to myoglobin but its limitations include a lack of complete cardiac specificity,a relatively small diagnostic window of24–30h after the acute event,and the probability of falsely increased values in patients with renal insufficiency cardiac specificity.H-FABP and myoglobin ratios have similar predictive accuracies for early detection of successful coronary reperfusion[67].Serial plasma H-FABP or myoglobin concentrations may be used for infarct sizing within the first24h after symptom onset only in AMI patients with normal renal function[68].Several immunosensors have been developed using enzyme amperometric,immuno-optical,or immunoassay technologies,as reviewed by Chan et al.[66].At present there is only one immunochromatographic POCT assay248Coronary Artery Disease2010,Vol21No4for H-FABP(rennesens CardioDetect)available commer-cially.The use of H-FABP in ruling out MI in patients with ACS is promising but needs further study. Phospholipase enzymes and cholineThe idea of a marker of plaque destabilization for the very early diagnosis of patients with MI is very attractive. Here,the marker is effectively a surrogate for direct detection of ischemia.Plaque rupture/erosion is the underlying pathophysiological event,which will lead to ischemia then to infarction.A plaque rupture marker does not have the problems of need for a gold standard to diagnose ischemia as hard outcomes such as death, AMI,and major adverse cardiac events(death,AMI, readmission with UA,and need for urgent revasculariza-tion)can be used as endpoints.A plaque rupture marker may still have the problem of cardiospecificity.There is a significant interest in the possibility that choline and phospholipase A2and D may be good markers of plaque instability.Phospholipases are enzymes subgrouped into four major categories(A–D)that catalyze phospho-lipids into fatty acids and another lipophilic substance. Lipoprotein-associated phospholipase A2,also known as platelet-activating factor acetylhydrolase,is regulated by mediators of inflammation.It circulates bound mainly to LDL and HDL and has been found to correlate with the levels of LDL,another indicator of CAD[69].In one study lipoprotein-associated phospholipase A2was associated with an almost two-fold increase in stroke in the selected population coupled with a six-fold increase in hyperten-sive individuals,leading to interest in its assay[70]. Phospholipase D catalyzes membrane-bound phospho-lipids producing phosphatidic acid and choline.It is also involved with the promotion of fibrinogen binding to platelets[71].Several experimental studies support the concept that phospholipase D activation is a key event in early ischemic membrane damage and in coronary plaque destabilization.Choline has been identified as a pro-mising marker for ACS by metabolic screening of human blood[72].Choline appears to be useful for risk stratifications in patients with angina pectoris,particu-larly if tests for troponins are negative on admission. Increased levels of plasma and whole blood choline concentrations are observed in tissue ischemia in patients with negative troponin values.Choline is not a marker for myocardial necrosis but indicated high-risk UA in patients without AMI(sensitivity86.4%,specificity86.2%)[73]. Assessment of both increased levels of plasma and whole blood choline may prove to be useful in patients suspected of ACS.At present,reliable choline assays are based on high-performance liquid chromatography–mass spectrometry, but development of rapid central laboratory assays and POCT for choline is expected to help to identify such high-risk patients in the emergency room as well.Current value of ischemia markersAlthough markers for ischemia are not specific to cardiovascular disease,further clinical studies are war-ranted so that this exciting tool may be used to routinely detect MI before myocardial necrosis.At present,IMA’s role is limited to ruling out ischemia rather than as a diagnostic test for the occurrence of ischemia because of its lack of specificity.Increased GPBB is specific for ischemic myocardial injury and has shown potential in assessing the effectiveness of thrombolytic therapy and in early diagnosis of ACS;however,a quick automated assay is not yet widely available.POCT has been developed both for FFA and H-FABP.FFA promises to be an early indicator of myocardial ischemia and H-FABP’s role in diagnosing AMI has been found to be comparable with myoglobin.Early markers for cardiac tissue necrosisMyoglobinMyoglobin is a heme protein that is found in all striated muscle fibers,which accounts for about2%of both skeletal and cardiac tissue mass(Fig.2).The small molecular weight of myoglobin allows it to be rapidly released during skeletal muscle injury and cardiac tissue necrosis[74].This is unlike creatine kinase and its isoform CK-MB,which are larger molecules and are released more slowly after an AMI[75].Myoglobin typically rises2–4h after onset of infarction,peaks at 6–12h,and returns to normal within24–36h(Fig.2).In one study measurement of myoglobin levels in blood samples obtained2h after the patient came to the ED with chest pain had a sensitivity of100%for detecting AMI[76].Myoglobin is released in a pulsatile manner,so serial testing with early presentation may be needed [77].Myoglobin has an excellent NPV(99.9vs.95%for CK-MB)if determined early enough to onset of symptoms,generally within the first hour[78].Rapid Fig.2100010010Hours after chest painRelativemarkerincrease10.1Early,intermediate,and late cardiac markers for necrosis.Cardiac biomarkers–the old and the new Singh et al.249。