Introduction

At one time, the term "cardiovascular risk" related to the identification of an individual experiencing an acute myocardial infarction (MI), with CK-MB serving as the laboratory's gold standard. In 2000, the European Society of Cardiology (ESC) and the American College of Cardiology (ACC) redefined the criteria for a myocardial infarction to include a typical pattern of increase followed by a gradual decrease in troponin T (cTnT) or I (cTnI) as the preferred biomarker due to its greater specificity and sensitivity relative to CK-MB values (mass assay).^[1] The ESC/ACC guidelines suggested that in the absence of troponin values, CK-MB serve as a substitute marker. In addition to these biomarker changes, the guidelines recommended that at least one of the following must also be present: (1) ischemic symptoms; (2) electrocardiogram (ECG) changes consistent with either a(n) (a) infarction, (b) ischemia (pathologic Q waves, ST-elevation/depression, or non-ST-segment elevation (NSTEMI), or (c) previous coronary artery intervention; or (3) pathologic findings of an acute MI (myocyte cell necrosis) including findings of a healed or healing MI. This expanded definition of a MI as more than a necrotic event led to significant changes in the identification and treatment of cardiac disease. In 2002, the ACC, in conjunction with the American Heart Association (AHA), produced a set of practice guidelines that defined the term acute coronary syndrome (ACS).^[2] The ACC/AHA document linked not only those patients experiencing a MI and the associated changes previously described by the ESC/ACC but also those who present with chief clinical complaints associated with unstable angina (chest pain, heartburn in conjunction with chest pain or tightness, dyspnea), heart failure and whose history includes diabetes, smoking, hypertension, cocaine use, hyperlipidemia, and family history (  ). The term ACS now serves as an operational term that encompasses a very heterogeneous patient population who present with any number of signs and symptoms associated with likely ischemic risk, including MI, unstable angina, and sudden cardiac death. Causes of ACS relate to either an imbalance caused by a reduction in the supply of oxygen to the myocardium, often linked to coronary artery disease (CAD)/cardiovascular disease (CVD), and/or an increased myocardial demand. The primary objective becomes early diagnosis allowing appropriate treatment in patients suspected of having ACS and the prevention of adverse outcomes. From a public health viewpoint, preventative care must focus on minimizing the development of CAD and the conditions leading to ACS and its more severe consequences including heart failure. In 2005, the direct and indirect cost of cardiovascular disease is estimated to be $393.5 billion and remains the leading cause of death in the United States.^[3]

It is well established that atherosclerosis is an inflammatory disorder rather than a cholesterol/lipid storage issue.^[4] When vascular endothelium cells are "insulted" in some fashion, they become damaged. There are many mechanisms leading to cell injury which include bacterial infection, hyperlipidemia, vasoconstricting hormones associated with hypertension, products of glycosylation seen in diabetes, pro-inflammatory cytokines derived from adipose tissue in obese individuals, or exposure to other toxins, including environmental by-products of pollution such as second-hand smoke. As a result of this injury, the damaged cells secrete selectin molecules (p-, e-, and l-), adhesion molecules (intracellular and vascular cell-ICAMs and VCAMs, respectively), and selective chemokines causing monocyte and T lymphocytes to adhere to the endothelial surfaces. These cells penetrate the vessel walls taking up residence in the endothelial lining interacting with both the endothelial and smooth muscle cells that make up the arterial wall. Modified lipoproteins (notably oxidized low-density) binding to vascular matrix proteins (collagen and fibrinogen) may eventually become ingested by the macrophages, resulting in lipid-laden cells, known as foam cells. In conjunction with macrophage related changes, T cells that evolve into T_H1 species secrete pro-inflammatory cytokines (notably, interleukin-1, interferon-gamma, and tissue necrosis factor-alpha (TNF-a) further amplify the inflammatory response. T-cells activated via antigen receptors can also produce cytokines that can interact with the foam cells through various mechanisms, notably CD₁₅₄ on the surface of the T cell binds to the CD₄₀ ligand (sCD40L) located on the foam cell. The foam cells secrete their own pro-inflammatory cytokines and metalloproteinases (MMPs). The MMPs are proteolytic enzymes that are also stimulated by the CD₄₀ ligand previously encountered on the T cell surface. Any mast cells attracted to the vascular lumen that undergo degranulation release proteinases causing the inactive MMP zymogen to form MMPs, further amplifying the effect. These mast cells also release heparin, TNF-a, and chymase, involved in angiotensin activation. Additionally, substances within the extracellular matrix, such as proteoglycans, continue to bind lipoproteins that are now susceptible to modification. Products of lipoprotein oxidative modification and glycosylation, including oxidized phospholipids, help maintain the inflammatory response. As this process continues, fatty dots or streaks may be formed. A combination of the cells involved proliferates forming fibrous connective tissue that then encapsulates any residual lipids deposited by dying macrophages. Eventually, this cell combination gives rise to a complex extracellular matrix and formation of a fibrous cap. Development continuing over many decades eventually gives rise to plaques that are distributed throughout the vasculature. The plaques may become calcified or collagenized contributing to the rigidity of the vascular walls referred to as stenosis,^[5] or at one time, "hardening of the arteries."

If these conditions prevail and risk factors persist, the lipid core continues to grow, pushing the arterial wall outward, while MMPs degrade the extracellular matrix, thinning the fibrous cap, and making it susceptible to tears. Once ruptured, platelets activated by thrombin in the vascular system begin the coagulation cascade forming a thrombus, which now becomes an integral part of the plaque. Repetition of this process can lead to further plaque growth and repair. If the plaque fully occludes the artery, necrosis ensues, resulting in a major infarction. However, if the plaque partially occludes a vessel, the aberrant pathology may go undetected until such time that it produces symptoms of angina, often under conditions of increased cardiac demand. This may lead to unstable angina, cerebral or pulmonary infarction, and episodes of ischemia. Many lipid lowering medications decrease the inflammatory response via alterations in the lipid core and stabilize the plaque, making it less vulnerable to rupture and repeated thrombic processes.

Providing proper cardiac care and treatment, with an emphasis on biochemical markers falls into 2 categories: those substances that can accurately predict the severity of CVD as an aid in treatment of risk stratification in an acutely ill symptomatic population and those markers that can help assess current cardiovascular health or likelihood of future risk in the general outpatient population. Several candidate substances currently being assessed for potential use and adoption include those associated with the various stages of vascular inflammation, related ischemia, plaque vulnerability, necrosis, and heart failure.     

The diagnostic challenge is significant in light of the fact that both groups often share similar underlying etiologies and nonspecific clinical complaints at a time when analytical markers lack the needed diagnostic sensitivity and specificity to absolutely predict the correct outcome. Some of the most promising markers for ACS and cardiac health risk assessment provide overlapping information as both groups share common etiologies associated with evidence of vascular inflammation, ischemia, plaque/clot rupture, and/or diminished vascular capacity. Regardless of the biomarker category, it should be remembered that there is a difference between diagnosis and prognosis.

Since it is estimated that between 2% and 5% of patients with MI are missed and discharged,^[6,7] patients presenting with nonspecific chest discomfort to the emergency department, who lack other confounding risks of ACS, still require risk stratification. It is a delicate balance determining which patients are at significant cardiac risk from those who are not. These patients are often triaged to a chest pain unit where serial cardiac markers (eg, myoglobin, troponins, and CK-MB) are evaluated, and non-invasive testing helps rule out ACS. Still, some 4% to 14% of these patients with negative studies go on to have serious future outcomes.^[8,9] In a recent report reviewing the literature between January, 1966 and May, 2005, some 5,436 citations involving 56 markers were listed.^[10] The race is on.

Early Markers of Cardiac Biomarkers for Risk Stratification in Cases of Suspected ACS

Current early markers for necrosis in patients that present within 6 hours of the onset of symptoms include myoglobin and CK-MB isoforms.^[11] These markers increase to detectable levels within 1 to 3 hours of onset of pain and generally return to within the reference ranges in approximately 24 to 36 hours, respectively, if not sooner. Myoglobin, an oxygen carrying protein in muscles, is released in the presence of skeletal muscle injury as well as cardiac tissue necrosis. Accumulation of myoglobin, regardless of its source, is nephrotoxic and may lead to renal insufficiency, which also places the patient at increased cardiac risk. Myoglobin is released in a pulsatile fashion, so serial testing with early presentation may be needed.^[12] Myoglobin has an excellent negative predictive value (99.9% versus 95% for CK-MB) if determined early enough to onset of symptoms, generally within the first hour.^[13] CK-MB isoforms that are released during myocyte necrosis are not ordinarily present in the blood, and while the CK-MB₂ isoform demonstrates excellent specificity within 0 to 6 hours, it suffers from the same kinetic release dynamics as CK-MB isoenzyme.^[14] CK-MB₂ is detectable in serum within 2 to 4 hours after onset and peaks at approximately 6 to 9 hours. Positive results, particularly when isoform ratios (MB₂/MB₁) are used, can be seen in patients with muscular dystrophy, or severe skeletal muscle damage.^[15] The turnaround time for CK isoforms using high-voltage electrophoresis is relatively long (approximately 25 minutes) using a test platform that does not readily lend itself to point-of-care testing. This may be the reason it has not enjoyed more widespread utilization. Because of the drawbacks of both myoglobin and CK-MB isoforms, the search for an improved early marker continues.

Intermediate/Late Markers of Necrosis

Troponin T (cTnT) and I (cTnI), along with CK-MB (mass assay) are reliable markers of cardiac tissue necrosis. Troponin release is similar to that of CK-MB but have the added advantage of remaining elevated longer (~7 to 10 days) after a "classic" MI but a much lower increase and elevation duration in cases of "microinfarction."^[16] Unlike CK-MB which is released from a number of non-cardiac muscles, antibodies used in the current generation of cTnT and cTnI assays are highly specific for their respective cardiac isoforms for each of the troponin proteins. It should be noted that interferences due to increased levels of fibrinogen, related either to the patient's status or a property of the sample, and the presence of autoantibodies to cTnI have been reported.^[17] While assays for cTnT are currently only commercially available from one manufacturer, there are at least 15 different manufacturers for cTnI.^[18] This lack of standardization between cTnI assays means test results from different assays are not comparable leading to a debate regarding the application of cut-off points and their subsequent interpretation. Because of concern that "microinfarctions" produce much lower increases in troponin values, guidelines proposed by the ESC/ACC^[1] recommend that the cutoff be set at the 99th percentile of a reference population with the assay having a coefficient of variation less than 10%. The wide variations of assays and their associated cutoffs only compounds this issue as does the fact that few assays can attain that level of imprecision.^[19,20] Another issue relates to the fact that low levels of troponins are found in the reference population, but below the lowest level of assay detection. As the assays improve, further studies will be needed to determine the clinical significance, if any, of very low levels of troponins. The message that should be remembered is that elevations in cTnT and cTnI do indicate some type of cardiac distress but not necessarily the cause or mechanism of the injury since increased levels are associated with a number of clinical situations with or without ischemia.^[21,22] . In these situations, the clinician needs to identify if ACS is present requiring follow-up and treatment. Laboratories need to carefully establish their own reference ranges for the troponin assays used and to make the medical staff aware of their level of imprecision and the limitations to the interpretation of a given assay for the test population.

Heart Failure

Brain natriuretic peptide (BNP) and the terminal fragment of its prohormone (NT-pro BNP) are hormones released from cardiac tissue in response to ventricular wall stress in the absence of necrosis and preceding angina and ST-segment changes.^[23,24] Recent evidence suggests that the use of either marker may contribute to both the diagnosis and prognostic outcome in patients with a MI.^[25] The plasma concentration of BNP rises quickly, peaking at about 24 hours after infarction, with the level and duration of elevation corresponding to the likelihood of future adverse cardiac events.^[26] NT-proBNP has a longer biological half-life (~1 to 2 hours) than does the biologically active BNP (~20 minutes). Early studies examining populations of patients with dyspnea, demonstrated that BNP was a sensitive marker in the diagnosis of congestive heart failure in this subpopulation of patients having left ventricular dysfunction.^[27-29] Heart failure begins with some type of myocardial injury generally secondary to a MI, other ischemic events, arrhythmia/atrial fibrillation, and hypertension. Elevations in the natriuretic proteins are not specific for heart disease but merely an indication of hemodynamic stress and fluid overload states. These include any edematous conditions including those associated with renal, lung (eg, cor pulmonale, pulmonary hypertension, acute pulmonary embolism),^[30] and liver disease; plus situations in which there is a triggering of the renin-angiotensin system including any increases in aldosterone leading to hemodynamic stress. However, evaluation of BNP and NT-proBNP values show good correlation between other assessment criteria schemes used by physicians (eg, New York Heart Association functional class, Framingham score, and echocardiogram results) in assessing dyspnea and CHF^[31-33] and as good predictors of future adverse cardiac events.^[34] Natriuretic protein levels increase with age, gender (females), and in patients with sleep apnea.^[35] Lower levels of BNP, but not NT-proBNP, are seen in obese patients. The exact cause is not known, but it has been suggested that increased adipose tissue alters the clearance of BNP but not NT-proBNP.^[36] It is thought that NT-proBNP is primarily cleared by the kidneys so alterations in renal clearance impact the NT-proBNP levels causing an overestimation in patients with severe renal disease.^[37] In patients receiving exogenous BNP treatment (eg, nesiritide), BNP levels will be impacted but not NT-BNP levels.^[38] Hypertensive medications and dietary salt-intake might also influence the natriuretic hormone levels.

It has been found that BNP and NT-proBNP were equally useful when indicating increased intracardiac pressure and the presence of cardiac structural disease in asymptomatic patients having systemic arterial hypertension, regardless of the underlying pathology.^[39] Just as with troponin assays, quality specifications for BNP and NT-proBNP assays are not universally adopted, and any cutoff values applied for determining clinical significance need to reflect the biases associated with both the analytical aspects of the assay employed, along with the consideration of population pre-analytical factors (eg, age, sex, obesity, renal problems, sampling considerations, etc) that might influence the establishment of either the reference ranges or any cutoff utilized. Samples of BNP should be collected in plastic tubes containing EDTA unless the assay has been validated for collection in glass tubes, while NT-proBNP should use serum, and use of any anticoagulant should only occur after suitable direct comparison and validation with serum samples. Sample stability appears to be assay dependent and should also be validated. In the absence of this information, it is recommended that BNP be analyzed within 4 hours at room temperature or stored with suitable additives. It appears that NT-proBNP is stable for 72 hours at room temperature and at 4B0C.^[40]

Inflammatory Markers

The overlapping nature of cardiac disease and inflammation makes distinguishing a biomarker as strictly related to plaque integrity, platelet activations, related to ischemia, or inflammation difficult, if not impossible. However, as an attempt at classifying various markers, compounds are linked to their place in the process. C-reactive protein (CRP), a well-established early acute phase reactant reflecting some type of inflammatory process, has also found utility in assessing the presence and severity of ACS. Increases in CRP using assays with expanded sensitivity to very low levels of CRP, so called high sensitivity CRP (hs-CRP) demonstrated a strong correlation as an independent risk factor for future cardiac events. Ridker and Cook, using data from the participants in the Woman's Health Study (27,939 women health care professionals over 45 years of age), reported that hs-CRP values less than 0.5 mg/L might suggest not only decreased cardiovascular future risk but suggested that CRP may play a critical direct role in atherothrombosis and its absence may be protective.^[41] Higher levels (>10 mg/L) are consistent with an increased risk of a cardiovascular event and may reflect a silent inflammatory process in patients without evidence of other causes of inflammation. Guidelines published by the Centers for Disease Control/AHA indicate that based on results using standardized assays with precision down to or below 0.3 mg/L, cut points of low risk (<1.0 mg/L), average risk (1-3 mg/L), and high risk (>3.0 mg/L) be assigned to those patients with an intermediate 10-year CHD risk (10% to 20% Framingham Risk Score/ATP III guidelines). Also included in the recommendation is that at least 2 samples; 1 fasting and 1 non-fasting in the asymptomatic general population, be tested.^[42]

Data suggests that hs-CRP predicts new coronary events in patients with ACS and unstable angina, acute MI, and risk of restenosis after revascularization procedures, independent of troponin T.^[43] The predictive qualities of hs-CRP for these patients may benefit by the application of cutoffs that are different from those previously mentioned for the prediction of risk in asymptomatic patients. Elevated hs-CRP levels also seem to predict prognosis and recurrent events in patients with stroke^[44,45] and peripheral arterial disease.^[46] These data suggest that hs-CRP may have a role in risk stratification of patients with established CVD. Nawsad and colleagues, measuring baseline and CRP values the day following percutaneous coronary intervention (PCI), confirmed that CRP might be of prognostic value in risk stratification independent of the anticipated myocardial response to PCI.^[47] Use of acetylsalicylic acid in minimizing adverse cardiac outcomes was also associated with a lower baseline CRP and with a decreased incidence of death or nonfatal myocardial infarction during follow-up. While widely accepted, limitations to hs-CRP involve estimations that more than 30% of patients with severe unstable angina do not present with elevated hs-CRP levels.^[41]

Many of the new markers showing some promise are related to the inflammatory response. One of these is myeloperoxidase (MPO). Myeloperoxidase is a lysosomal enzyme, requiring heme as a cofactor, released from neutrophilic granules, monocytes, and some subtypes of tissue macrophages. Neutrophils engulf pathogens and use MPO, which catalyzes the reaction of hydrogen peroxide with chloride ions to produce a strongly antiseptic hypochlorite ion, a reactive oxygen species, to destroy them. Myeloperoxidase is also linked to oxidation of lipids in low-density lipoproteins (LDL), dysfunctional high density lipoproteins (HDL), and consumption of nitric oxide thereby rendering the normally anti-thrombotic endothelial surface thrombogenic via the expression of various pro-thrombotic and anti-fibrinolytic factors.^[48] Increased MPO levels may also be associated with adverse ventricular remodeling after myocardial infarction and changes associated with progression to congestive heart failure.^[49] Individuals with inherited low levels of MPO were found to be cardio-protected.^[50] As previously discussed, plaque destabilization is a result of both inflammatory mechanisms and those related to actual plaque development via foam cells. Myeloperoxidase is secreted by the macrophages along with MMPs. Myeloperoxidase plays a role in the degradation of the fibrous cap, making it both a marker of inflammation and one of plaque instability. Interest in MPO intensified after a report by Brennan and colleages^[51] indicated that a single initial measurement of plasma myeloperoxidase independently predicts the early risk of myocardial infarction, as well as the risk of major adverse cardiac events in the ensuing 30-day and 6-month periods. Myeloperoxidase levels, in contrast to troponin T, creatine kinase MB isoform, and CRP levels, identified patients at risk for cardiac events in the absence of myocardial necrosis, highlighting its potential usefulness for risk stratification among patients who present with chest pain. Men were found to have higher mean values as were individuals with hyperlipidemia. The link between MPO and adverse outcomes in chest pain patients with initially negative cTnT, has spurred development and relatively recent FDA approval (May 2005) of an enzyme-linked immunosorbent assay (CardioMPO, made by PrognostiX) for the quantitative determination of MPO in human plasma.

The MMPs, closely associated with mechanisms involved with MPO, are protease enzymes requiring zinc, produced by smooth muscle endothelium and monocytes. They are sub grouped based on substrate specificity and structure, with MMP-2 and -9, (gelatinases) being of most current interest in inflammation and cardiac disease. The MMPs are found in most tissues and are regulated by transcription in response to growth factors, cytokines, and hormones, and extracellularly, in the form of prohormones, whose breakdown occurs mostly in response to plasmin. Recall, that MMPs are also stimulated with the CD₄₀ receptor ligand. There are a group of specific endogenous tissue inhibitors of metalloproteinases, known as TIMPs, that regulate the effects of MMPs. Pharmacologic studies involving increasing TIMPs thereby minimizing the effects of MMPs are currently under investigation. Decreases in MMPs are associated with the renin-angiotensin system, aspirin, atorvastatin, and doxycyline.^[52] Thrombic complications are associated with arterial wall changes due to smooth muscle endothelial changes, contributing to plaque formation as well as plaque destabilization, and vascular remodeling after necrosis. Studies measuring MMP-9 seem to indicate that it may be another risk factor for assessing the severity of coronary artery disease in patients with ST-elevation MI.^[53] It has been hypothesized that estrogen may upregulate MMP-9, causing any existing atherogenic plaque in older women on hormone replacement therapy (HRT) to be at greater risk of CHD and adverse outcomes of ACS.^[54] This explanation may, in part, explain the apparent relationship between HRT and cardiac disease found in the Women's Health Initiative.^[55]

Because of its involvement in atherogenic and thrombic processes, sCD40L, which binds with the CD₄₀ receptor derived not only from T lymphocytes but other involved cells, may offer another alternative in ACS development in the early estimation of processes that may trigger plaque rupture associated with platelet activation. Increases in sCD40L have also been demonstrated in a number of different inflammatory processes, so it is not specific for cardiac inflammation. No commercial kit has been cleared by the FDA to date.

Two markers of recent interest relating to plaque vulnerability are pregnancy-associated protein A (PAPP-A) and placenta growth factor (P1GF). Pregnancy-associated protein A is a metalloproteinase, initially identified in the sera of pregnant women, and used in screening for Down's syndrome.^[56] Pregnancy-associated protein A is a large, zinc-binding proteinase produced by different cell types, including fibroblasts, vascular smooth muscle cells, and male and female reproductive tissues. Pregnancy-associated protein A is 1 of 6 different proteases that degrades insulin-like growth factor binding proteins (IGFBPs). This proteolytic degradation of the IGFBPs is considered the predominant mechanism for the release of bioactive insulin growth factor-1 (IGF-1).^[57] A large study has illustrated that decreases in IGF-1 appear 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.^[58] It is believed that PAPP-A is released during plaque destabilization and appears to be a valuable indicator of unstable angina and acute MI in patients lacking other indicators of necrosis.^[59] The apparent discrepancy between low levels of IGF-1 and PAPP-A brings into question the exact physiologic role that PAPP-A may play in plaque disruption.

Placenta growth factor, is a member of the vascular endothelial growth factor family, which stimulates vascular smooth muscle cell growth, recruits macrophages into atherosclerotic lesions, up-regulates production of tumor necrosis factor-a and monocyte chemotactic protein 1 by macrophages, and stimulates pathological angiogenesis.^[60] It appears to be an initiator of the inflammatory process. In one study, elevated 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.^[61] Additional trials are underway to verify these findings.

Markers of Ischemia

To date, only ischemia modified albumin (IMA) is approved by the FDA, using the albumin cobalt binding test (ACB), for assessment of myocardial ischemia. It was determined that an alteration in the N-terminus end of human serum albumin (HSA) occurs to a greater extent in patients experiencing ischemia. This damage is most likely due to damage caused by oxidative free radicals prevalent during ischemic events, and as a result, HSA demonstrates altered binding of trace metals resulting in IMA. There are 2 forms of IMA one in which HSA binds mostly copper, and a second form in which the damage to the N-terminus prevents metal binding. Patients without ischemia have more available metal binding sites on their HSA, than those from ischemic patients. The FDA approved method for IMA (ACB Test by Ischemia Technologies) uses cobalt in its assay; no immunoassay has yet been developed. When cobalt (in vitro only) is added to a sample, normal HSA will bind it, leaving little residual cobalt while any IMA present, due to its altered binding site, cannot. Increased amounts of IMA, as demonstrated in patients having transient ischemic episodes following PCI without concurrent myocyte damage,^[62] result in less cobalt binding and more residual unbound cobalt available for complex with a chromogen (dithiothreitol) which can be measured photometrically. An increase in IMA is inversely related to the amount of cobalt causing an increase in colored product produced in the test platform. It is estimated that approximately 1% to 2% of the total albumin concentration in the normal population is IMA compared to 6% to 8% in patients experiencing ischemia. The clinical utility of IMA appears to be as a negative predictor for ischemia and ACS, particularly when used in conjunction with other tests. While the optimum cutoff for IMA for ruling out ACS is 85kU/L, the manufacturer has suggested a higher value of 100 kU/L for risk stratification. There also exists an overlap between the normal population and that of individuals with cardiac ischemia. Ischemia modified albumin is not specific for cardiac ischemia. Ischemia modified albumin is also elevated in most patients with cirrhosis, bacterial and viral infections, advanced cancers, stroke (brain ischemia), and end-stage renal disease. It does not appear that IMA is significantly elevated in patients with autoimmune diseases, benign gastrointestinal disease, orthopedic injuries, or non-ischemic cardiac conditions. Data exists suggesting that there is a decrease in IMA associated with muscle ischemia related to increased lactate production and a shift in albumin levels following strenuous exercise requiring possible consideration when interpreting IMA in certain populations, including those with peripheral vascular disease and in marathon runners.^[63] Additional data are needed to clarify the impact alterations in total albumin values may have on IMA interpretation. While a promising marker in certain clinical situations, the number of possible interferences may limit its utility for patients suspected of ACS.

Evidence exists that unbound free fatty acids (FFA_u) increase significantly in ischemic-related events.^[64] Fatty acids are essential building blocks for many lipids and are used for energy production during times of fasting or increased metabolic demand. Fatty acids are either esterified (bound to glycerol or other alcohol), non-esterified but bound to albumin (FFA), or to a much smaller extent, present in an unbound soluble form (FFA_u).^[50] It is not fully understood what role FFA_u plays in cardiac disease possibly participating in the developing necrotic process and released as a result of cell rupture or other precipitating conditions, or serving as an activator with other molecules involved in ischemia. Currently, a fluorescent probe assay is available.

Transport across membranes is enhanced by fatty acid binding proteins (FABPs). Nine different FABP isoforms have been identified with a predictable tissue distribution and fairly long half-life of several days.^[65] Concentrations of these FABPs are not tissue specific, but heart-type FABP (H-FABP) is released after cardiomyocyte damage within 6 hours, similar to that of myoglobin. It has been found that H-FABP may perform better and reach its upper reference limit sooner than either myoglobin or troponin.^[66] Heart-type FABP is not cardiac specific but is also released to a smaller extent in skeletal muscle, distal tubular cells of the kidney, specific parts of the brain, lactating mammary glands, and the placenta.^[67] A number of enzyme immunoassays are available for H-FABP testing. Considered as a necrotic marker, its association to ischemia and prognosis for adverse events may be elucidated.

Phospholipase enzymes A2 and D have sparked interest in their role in assessing ischemia associated ACS. Phospholipases are enzymes subgrouped into 4 major categories (A-D) that catalyze phospholipids into fatty acids and another lipophilic substance. Lipoprotein-associated phospholipase A₂(Lp-PLA₂), 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.^[68] A recent study showed that the total plasma Lp-PLA₂ activity_, was able to predict the presence of the more atherogenic small dense LDL particles, but that increased triglyceride levels are a better predictor in individuals with hyperlipemia.^[69] Ironically, there has been a renewed interest in this assay, not for use in cardiac assessment as it was originally approved by the FDA, but rather in stroke prediction after it was found that elevated levels of Lp-PLA2 were associated with an almost 2-fold increase in stroke in the selected population coupled with a 6-fold increase in hypertensive individuals.^[70]

Phospholipase D (PLD) catalyzes membrane bound phospholipids producing phosphatidic acid and choline. It is also involved with the promotion of fibrinogen binding to platelets.^[71] Increased levels of plasma (PLCHO) and whole blood choline (WBCHO) concentrations are observed in tissue ischemia in patients with negative troponin values. Choline was not a marker for myocardial necrosis but indicated high-risk unstable angina in patients without acute myocardial infarction (sensitivity 86.4%, specificity 86.2%).^[72] Assessment of both plasma PLCHO and WBCHO may prove to be useful in patients suspected of ACS.

Each of the markers discussed in this review has limitations. An ideal marker is one in which there is a specific easily measurable increase that clearly aligns with a predictable outcome be it evidence of ischemia, inflammation, myocardial necrosis, plaque rupture, plaque destabilization, or heart failure. It should reflect effective treatment or prediction of adverse outcomes, and readily defined as either a marker rather than a risk factor. That is, does the analyte change as a result of the process (marker) or predictor of future events (risk factor)? The assay should be cost-effective, easy to perform, having a relatively short turnaround time. The appeal and popularity of some markers rests on their point-of-care platform, with more being proposed, including a lab-on-a-chip assay offering both CRP and leukocyte counts.^[73] Discovering that more familiar markers, such as serum g-glutamyltransferase at levels within the physiologic range, once thought unrelated, may have some relevance in detecting oxidative stress associated with ACS, adds valuable insight into the genesis of vascular disease.^[74] Because of the underlying shared etiologies related to the process of arteriosclerosis and the complexity of the pathological processes giving rise to adverse thrombic outcomes, a single marker that relates to each stage is unlikely. Use of multiple markers with varying decision levels to either rule-in or rule-out a clinical decision is more probable. Many algorithms and intelligent electronic approaches are currently being investigated. Having markers meet the needs of both emergency room physicians triaging chest pain patients and cardiologists examining a subset of CHD patients provides another layer of complexity. As the understanding of the intricacies and interrelationships between ACS, inflammation, and other disease processes develops, so does the number of possible tests, and the ongoing search for the holy grail of cardiac markers will continue.