The quest for a biochemical marker of myocardial ischemia

Finding a sensitive and specific biochemical marker to demonstrate ischemia has been elusive. Specific candidate biomarkers are discussed in detail in Chapter 13. Before considering the evaluation of these markers, a broader overview of the approaches that have been taken in the pursuit of markers of ischemia is useful.

Approaches Related to Reactive Oxygen Species

Reactive Oxygen Species in Atherothrombosis

Ischemia can result in tissue damage through the formation of a number of injurious compounds. A significant body of research over the past 15-20 yr has focused on the role of reactive oxygen species (ROS), including oxygen free radicals, singlet oxygen, and peroxides. The tissue damage induced by such compounds is well described and appears to be an ubiquitous biological phenomenon (Fig. 4). The alterations that occur to proteins, lipids, nucleic acids, membranes, and so forth are relatively independent of the tissue involved. However, the propensity to generate any specific ROS may differ among tissues and even between species for the same tissue. Furthermore, natural protective measures, such as catalase and superoxide dismutase, also vary in their distribution among tissues and show significant interspecies variations. Both the inherent propensity to generate a specific profile of ROS under given pathophysiological conditions and the intrinsic protective pathways will ultimately determine the response to ischemia. This is an important point because it raises challenges to studying animal models in which to develop new markers for detecting human disease.

The role of ROS in atherogenesis has been well documented, in particular through the oxidation oflow-density lipoprotein (LDL) (33). Holvoet et al. (34) demonstrated that there was an increase in a specific oxidized species of LDL (malondialdehyde [MDA]-modified LDL) in patients with ACS. Of note, both AMI and unstable angina showed similar elevations in MDA-modified LDL, and subsequent analyses have suggested that this may not be an acute marker ofischemia but, rather, reflects an underlying state linked to the development of coronary atherosclerosis. This difference raises an important consideration for bio-marker development, because markers that are specific to the disease state ofatherosclerosis may not be useful for the detection of acute events such as ischemia. Following the temporal changes in biomarkers during evaluation (i.e., a rise and fall in relation to the ischemic event) is likely to be useful in distinguishing those that may be viable as event markers.

There are novel biochemical pathways identified that become activated after exposure of the myocardium to brief periods of ischemia, allowing cells to better tolerate subse-

Fig. 4. Mechanism of oxidative tissue damage by ROS. Ischemia and the reperfusion that follows can result in the formation offree radicals and other ROS, including peroxides. All are capable of damaging tissues, and many biological safeguards at the cellular level have evolved to protect against oxidant damage. Ischemia/reperfusion can also be protective, in that short periods of ischemia followed by reperfusion can invoke a "preconditioning process" (see text) that affords cells protection against subsequent ischemic damage.

Fig. 4. Mechanism of oxidative tissue damage by ROS. Ischemia and the reperfusion that follows can result in the formation offree radicals and other ROS, including peroxides. All are capable of damaging tissues, and many biological safeguards at the cellular level have evolved to protect against oxidant damage. Ischemia/reperfusion can also be protective, in that short periods of ischemia followed by reperfusion can invoke a "preconditioning process" (see text) that affords cells protection against subsequent ischemic damage.

quent periods of ischemia. This so-called ischemic preconditioning appears to be related, at least in part, to the generation of ROS. Preconditioning occurs in two phases: one rapid in onset that lasts for several hours and one delayed that requires transcription and translation of specific genes appearing in 18-24 h and persisting for several days (35). The phenomenon of preconditioning provides a clear demonstration that ischemia can effect fundamental biochemical alterations, specific to cardiac myocytes, and suggests that there may be biochemical targets specific to the myocardium that can be used to detect ischemia.

Ischemia-Modified Albumin

To date, only one biochemical marker of ischemia has been approved by the Food and Drug Administration for the risk stratification of patients with suspected ACS. Nascent human serum albumin has multiple functions, including the ability to bind heavy metals at the N-terminal portion of the protein. The observations were made that not all albumin molecules have this metal-binding capability (36) and that in the presence of myocardial ischemia albumin may become altered; hence, this type of albumin was named ischemia-modified albumin (IMA) (Ischemia Technologies, Denver, CO) (37). The relative proportion oflMA increases significantly following the onset of myocardial ischemia and persists for approx 6-8 h (38,39). The initial hypothesis linked this change to ROS-induced damage to the N-terminus of albumin, which inactivated the heavy metal-binding site (Fig. 5). IMA is discussed in further detail in Chapter 13.

Myocardial Oxidative Metabolism

Technetium-Based Myocardial Imaging

Cardiac specificity is vital for the decision-making process in the management of ACS and, thus, there is a need to identify biochemical alterations specific to the myocardium

aibumin Oxygen Species Albumin

Fig. 5. Ischemic modification to albumin: free-radical damage to N-terminus. Among the myriad functions for human serum albumin is the binding of a number ofplasma constituents, including fatty acids (FA) and heavy metals such as copper (Cu++). There are a number of binding sites for the former, but the N-terminus appears to have a specific affinity for metal binding, which is susceptible to oxi-dant-induced damage. One proposed mechanism is that ROS such as hydroxyl radical COH) results in cleavage of a terminal peptide that abolishes the metal-binding capacity. This can be assayed by determining the binding capacity for cobalt, such as with the albumin-cobalt binding test, for detection of IMA.

Native Reactive Ischemia Modified aibumin Oxygen Species Albumin

Fig. 5. Ischemic modification to albumin: free-radical damage to N-terminus. Among the myriad functions for human serum albumin is the binding of a number ofplasma constituents, including fatty acids (FA) and heavy metals such as copper (Cu++). There are a number of binding sites for the former, but the N-terminus appears to have a specific affinity for metal binding, which is susceptible to oxi-dant-induced damage. One proposed mechanism is that ROS such as hydroxyl radical COH) results in cleavage of a terminal peptide that abolishes the metal-binding capacity. This can be assayed by determining the binding capacity for cobalt, such as with the albumin-cobalt binding test, for detection of IMA.

(or coronary vasculature). Technetium-based myocardial perfusion agents may demonstrate one such alteration. Although used for imaging, these agents are functionally biochemical markers ofischemia: they require active transport at the plasma and mitochondrial membranes, and they require a functional electron-transport system in order to be bound. These agents thus reflect the status of oxidative metabolism in the cardiac myocyte. The information provided has very powerful diagnostic and prognostic value (Fig. 6) (27). Thus, using technetium-based MPI, we have identified a potential biochemical target— the status of oxidative metabolism—through which to document myocardial ischemia. However, the detector for this biochemical event is not an antibody or a colorimetric assay that can be used in a simple point-of-care device but, rather, a large, expensive scinti-graphic camera requiring a significant level of computer processing and skill for interpretation. The potential advantages of developing a simple laboratory test to detect these biochemical events are obvious.

In addition, a potential for discordance between perfusion and functional imaging exists by virtue of the timing of these aspects of the test. One advantage to technetium-based agents is that their rapid uptake and retention allows perfusion images to be acquired several hours after injection, yet still reflect the perfusion status at the time of injection. Gated images, on the other hand, are acquired in real time and, thus, long delays between injection and imaging allow the possibility of finding abnormal perfusion with recovered wall motion. A similar discordance has been observed between pure perfusion imaging with thallium and metabolic imaging with iodine-123-labeled fatty acid analog, 123I-15 (p-iodophenyl)-3-(R,<S)-methylpentadecanoic acid (BMIPP). BMIPP is taken up in proportion to blood flow (40) but is also dependent on intracellular fatty acid metabolism (41). The molecular structure of BMIPP prevents metabolism via ^-oxidation but the vast majority is incorporated into the lipid pool via activation to BMIPP-CoA, which reflects intracellular adenosine triphosphate concentration. If ^-oxidation is impaired, such as in the setting of ischemia, then the tracer is not converted and diffuses back out of the cell. There is a well-described delay in the recovery of oxidative metabolism that can last several hours to days. This "ischemic memory" is thought to be a correlate of myocardial stunning

Fig. 6. Event rates categorized by clinical risk vs MPI. Primary risk stratification levels 2 (true unstable angina), 3 (probable unstable angina—higher risk nondiagnostic), and 4 (possible unstable angina—lower risk nondiagnostic) have decreasing event rates. The rates of AMI and revascularization are highest in level 2 patients relative to those in the lower-risk nondiagnostic levels 3 and 4. Secondary risk stratification using rest MPI is employed for level 3 and 4 patients (15 and 50%, respectively, of the chest pain population) to identify patients who are in fact at higher risk than appears from the initial risk stratification. An abnormal (Pos) MPI clearly identifies higher-risk individuals among this population, whereas a negative (Neg) MPI indicates very low risk. Solid bars represent AMI and hatched bars revascularization. Note that the total event rate associated with an abnormal MPI in level 3 and 4 patients is similar to the event rate for level 2. Thus, for patients who appear to be at lower risk based on history, physical examination, and ECG, a positive perfusion study carries equal probability of MI or urgent revascularization as does an ischemic ECG or other high-risk clinical featureson the initial evaluation (27).

Fig. 6. Event rates categorized by clinical risk vs MPI. Primary risk stratification levels 2 (true unstable angina), 3 (probable unstable angina—higher risk nondiagnostic), and 4 (possible unstable angina—lower risk nondiagnostic) have decreasing event rates. The rates of AMI and revascularization are highest in level 2 patients relative to those in the lower-risk nondiagnostic levels 3 and 4. Secondary risk stratification using rest MPI is employed for level 3 and 4 patients (15 and 50%, respectively, of the chest pain population) to identify patients who are in fact at higher risk than appears from the initial risk stratification. An abnormal (Pos) MPI clearly identifies higher-risk individuals among this population, whereas a negative (Neg) MPI indicates very low risk. Solid bars represent AMI and hatched bars revascularization. Note that the total event rate associated with an abnormal MPI in level 3 and 4 patients is similar to the event rate for level 2. Thus, for patients who appear to be at lower risk based on history, physical examination, and ECG, a positive perfusion study carries equal probability of MI or urgent revascularization as does an ischemic ECG or other high-risk clinical featureson the initial evaluation (27).

(42) and may prove valuable in the evaluation of chest pain (43). Thus, the ability to assess metabolism with the technetium agents and specific alterations in oxidative metabolism with BMIPP provides another avenue to detect myocardial ischemia. Can this metabolic alteration be assessed more simply using conventional biomarker technologies? The answer lies in the biochemistry of myocardial energetics.

Unbound Free Fatty Acids

The metabolic perturbations that occur during ischemia may suggest targets for marker development (Fig. 7). One such example is the detection of serum unbound free fatty acids (uFFA) by the high-affinity probe ADIFAB™, an acrylodated intestinal fatty acid-binding protein (FABP). The premise is that under normal circumstances serum albumin can essentially act as a buffer to sequester any uFFA not attached to FABPs, but under ischemic conditions there is an increase in FFA release, this buffering capacity is overwhelmed, and uFFA increases. Kleinfeld et al. (44) demonstrated a mean 14-fold increase following ischemia provoked by angioplasty, with the largest elevations occurring in those who also exhibited ECG changes during balloon inflation. Fatty acid accumulation may be owing to impaired utilization and uptake of FFA by the ischemic myocardium but may also occur through peripheral lipolysis activated indirectly secondary to ischemia, i.e., catecholamine release. It will certainly be interesting to determine whether there is a common relationship between the FFA and IMA models of ischemia detection.

Fig. 7. Simplistic overview of metabolic alterations in ischemic myocytes. Under oxidative conditions myocytes generate approx 70% of their energy via mitochondrial (M) P-oxidation of long-chain fatty acids (FA). Under hypoxic conditions, P-oxidation is inhibited and myocytes convert to a glucose-based metabolism. FAs that are normally carried by specific binding proteins (FABP) begin to accumulate and can be damaging to the cell if not somehow sequestered. Whether directly via a cardiac source or through some peripheral release mechanisms, FAs accumulate in the circulation. Initially they will bind to FABPs and to albumin, but when these sites are saturated, there will be an increase in unbound component, which can be detected quantitatively by the probe ADIFAB.

Fig. 7. Simplistic overview of metabolic alterations in ischemic myocytes. Under oxidative conditions myocytes generate approx 70% of their energy via mitochondrial (M) P-oxidation of long-chain fatty acids (FA). Under hypoxic conditions, P-oxidation is inhibited and myocytes convert to a glucose-based metabolism. FAs that are normally carried by specific binding proteins (FABP) begin to accumulate and can be damaging to the cell if not somehow sequestered. Whether directly via a cardiac source or through some peripheral release mechanisms, FAs accumulate in the circulation. Initially they will bind to FABPs and to albumin, but when these sites are saturated, there will be an increase in unbound component, which can be detected quantitatively by the probe ADIFAB.

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