Abstract
Ischemia-modified albumin (IMA) is assumed to be an “N-terminal modified” albumin that is generated immediately after myocardial ischemia. The diagnosis of AMI is based on the reduction in the binding affinity of cobalt for albumin, which is mainly attributed to the inability of cobalt to bind to the modified N-term of albumin. Although the albumin-cobalt binding test was accepted as a potentially powerful marker to discriminate acute coronary syndrome from non-ischemic chest pain, its usefulness has been questioned in recent years. Patients with acute ischemic myocardium have a rapid increase in serum levels of fatty acids (FA).
Almost all released FAs bind strongly to albumin, creating conformational changes in the protein with a consequent reduction in cobalt-binding affinity. There is a clear metabolic and temporal relationship between AMI measured by the albumin-cobalt binding test and serum AG levels. In line with what has recently been suggested in the literature, we conclude that a change from the concept of albumin from “N-terminal modified” to “occupied by AF” is required, since it better describes IMA in patients with acute coronary syndrome. We also offer “oxidation-modified albumin, AOM”, which is conceptually different from “FA-occupied” IMA, to describe the modification of albumin in chronic diseases associated with increased oxidative stress.
1. Introduction
Acute coronary syndrome is diagnosed biochemically by measuring serum myocardial proteins originally found in the cytoplasm, which appear in the blood no earlier than 4 to 6 hours after the rupture of the myocardial cell membrane. These proteins include creatine kinase MB (CK-MB) and troponin. Therefore, biochemical markers that are sensitive and/or specific to ischemia prior to cell damage are of great clinical importance. Bar-Or proposed such a serum-based biochemical test.
The basic principle of this test involves the N-terminal region of human albumin and its inherent affinity for the cobalt metal ion (the so-called albumin-cobalt binding, or ACB assay), the premise is that during myocardial ischemia, the Binding of albumin to cobalt affinity is reduced due to an N-terminal modification of albumin. Note that N-terminal modified albumin has also been referred to as ischemia modified albumin (IMA) since the first description of Bar-Or.
2. Cobalt Albumin Binding Assay (ACB)
The ACB trial was approved by the FDA in 2003 as a method to identify myocardial ischemia in patients admitted to the emergency department. In essence, the test involves adding cobalt chloride (approximately 1.5 equivalents per albumin molecule) to a serum sample, mixing gently, and then incubating to allow the albumin to bind to cobalt.
Dithiothreitol (DTT: a cobalt chelator) is added as a coloring agent, and the brown color produced by DTT-cobalt chelation (free or unbound) is measured at 470 nm using a spectrophotometer. A serum cobalt blank without DTT is used for comparison, and results are reported in absorbance units (ABSU). The ABSU data provides a measure of the concentration of chelated cobalt (free or unbound) in the sample and indirectly reflects the level of IMA; that is, albumin that is unable to bind to cobalt due to what is known as an “N-terminal modification.”
Although the ACB trial has had FDA approval for more than a decade, the trial has not achieved the expected clinical success. We have conducted a literature review of reports accumulated during the 15 years after the first description of the concept of “N-terminal modification” of albumin to investigate reasons for the limited precision and reproducibility of the ACB assay. We then proceed to describe a new concept in light of this knowledge.
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3. Interaction between fatty acids and albumin
Human serum contains a mixture of at least six FAs, as follows (with approximate percentages): oleic acid (38%), palmitic acid (25%), linoleic acid (22%), stearic acid (10%), arachidonic acid (3%) and linolenic acid (2%). Almost all blood FAs are strongly bound to albumin and are transported throughout the body in this form, while only a very small percentage (less than 1 / 100,000) are present in the free form, the so-called free or free FAs. Human serum albumin has at least seven binding sites, with varying affinities for medium and long-chain FAs.
Under normal physiological conditions, an average of 0.1 to 2 molecules of FA bind to each molecule of albumin; however, the molar ratio of FA to albumin can reach up to 6–7 during fasting or extreme exercise or in patients with liver and cardiovascular disease (eg, acute myocardial ischemia). The numbering of these seven sites (FA sites 1-7) in the crystal structure of albumin is arbitrary and is not based on affinity for FA molecules.
Among the seven binding sites on albumin, the FA2, FA4, and FA5 sites have been identified as having the highest affinity for FAs. Although the binding pockets appear to be well adapted to accommodate FA molecules, they are not specific to any particular FA and are therefore capable of binding to other ligand molecules. Fujiwara and Amisaki have recently summarized the list of competing ligands that share a common binding site with FAs.
4. Fatty acid as a marker of myocardial ischemia
Myocardial ischemia leads to a hyperadrenergic state a few minutes after the appearance of chest pain, which leads to the degradation of tissue and plasma phospholipids, as well as triglycerides, which increases the plasma concentration of free FA. Patients with acute ischemic myocardium have a rapid increase in serum-free AG levels, which may exceed normal mean values at admission by a factor of 3 to 10. The measurement of serum-free FA levels in the diagnosis of acute myocardial ischemia has been the subject of several patent applications.
Free FA serum levels have also been reported to increase within 30 minutes after coronary balloon angioplasty (a well-known in vivo model of transient myocardial ischemia caused by balloon inflation) and a mean increase in 5 times at AF levels. A recent multicenter study investigated the utility of measuring free FA levels compared to other available clinical tests (i.e. pro-B-type amino-terminal natriuretic peptide, IMA, heart fatty acid-binding protein, classic troponin T, and high-sensitivity troponin I) and found that free FA had the highest overall sensitivity (75%), specificity (72%), and negative predictive values (92%) for discriminating acute coronary syndrome from non-ischemic chest pain in patients admitted to the emergency department.
Therefore, current data, although limited, suggest that monitoring of free AF levels in patients presenting with chest pain may provide an early indication of myocardial ischemia capable of discriminating between acute coronary syndrome and non-ischemic chest pain.
6. Why should we make changes to the IMA concept?
As previously discussed, there is no positive correlation between the ELISA test developed specifically to detect the N-terminal modification of albumin and the classic ACB test; therefore, the N-terminal site of albumin is unimportant with respect to the results of the ACB assay for patients with the suspected acute coronary syndrome.
Furthermore, if we assume that the AMI is “AF-occupied” albumin rather than “N-terminal modified” albumin in patients with acute myocardial ischemia, then we conclude that the AMI measured by an ELISA assay is useless in discriminating acute coronary syndrome from the non-ischemic chest. pain. Investigators dealing with AMI-guided diagnosis of acute myocardial ischemia should keep this in mind and continue to use the classic ACB assay rather than an AMI ELISA that is not actually “N-terminal modified” but “busy. please”. albumin.
Researchers dealing with chronic diseases associated with “glycated/oxidized” albumin can continue to use both the classic ACB assay and ELISA tests to determine AMI levels, as irreversible modification of the N-terminal end (as well as many other parts of albumin, including cobalt binding sites) is common in such circumstances, which makes the ACB and ELISA tests useful.
Alzheimer’s disease is one of those well-defined examples of chronic disease for which long-term oxidative stress is important in pathogenesis, leading to increased protein oxidation, DNA oxidation, lipid peroxidation, end products of advanced glycation and carbonylated proteins. As expected, AMI levels measured by both the classic ACB assay and ELISA were significantly higher in Alzheimer’s patients and correlated well with other markers of oxidative stress.
A marker such as IMA that is sensitive and/or specific to myocardial ischemia prior to cell damage can be of enormous value to the emergency physician evaluating patients with chest pain. However, we still need a better understanding of this marker before it is ready for primetime use. All researchers interested in IMA should know what exactly IMA is, as well as the difference between the results of the ACB test and the ELISA test, and what the test results actually mean. Therefore, we must review the concept of IMA.
7. Conclusion
Despite its shortcomings, the ACB assay for AMI levels may be useful in emergency situations to discriminate against acute coronary syndrome; however, the ELISA test should not be used for this purpose. We conclude that a conceptual change from “N-terminal modified” to “AF-occupied” albumin is required to better delineate the IMA in patients with acute coronary syndrome. In this regard, we would like to offer the use of the new term “oxidation-modified albumin” (AOM) instead of the well-known “ischemia-modified albumin” to better delineate modified albumin in patients with chronic diseases associated with increased oxidative stress.
Therefore, the new nomenclature may be useful to differentiate the modification of albumin that occurs in acute coronary syndrome with myocardial ischemia from chronic disease with increased oxidative stress. Myocardial ischemia generates “ischemia-modified albumin, AMI” “reversible” secondary to the occupation of albumin by FA, while oxidative stress generates “albumin modified by oxidation, AOM” “irreversible” secondary to oxidation adducts on albumin.