A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia—a preliminary report¹

Abstract

We initially observed a phenomenon of reduced in vitro binding of exogenous cobalt [Co(II)] to the N-terminus of human serum albumin (HSA) in emergency chest pain patients with early onset unstable angina and myocardial infarction. We then developed a colorimetric assay to measure cobalt-HSA binding and record the results in absorbance units (ABSU). In a preliminary clinical study of 139 emergency patients with acute chest pain, 99 patients with evidence of myocardial ischemia (Group 1) had elevated assay levels (mean ABSU ± SD; 0.519 ± 0.086) compared to 40 patients (Group 2) with no evidence of ischemia (0.316 ± 0.092) (p < 0.00001). In Group 1, 95 of 99 (96.0%) patients had levels higher than a decision threshold of 0.400 ABSU and in Group 2, 37 of 40 (92.5%) samples had higher cobalt binding capacity (ABSU ≤ 0.400). Further studies are warranted to determine if an assay measuring altered cobalt-HSA binding is a clinically useful diagnostic test to rule out myocardial ischemia.

Introduction

The diagnosis of acute myocardial ischemia in emergency patients with acute coronary symptoms is often difficult due to an unclear clinical presentation and lack of a rapid, reliable diagnostic test. Resting electrocardiogram (EKG), creatine phosphokinase isoenzymes (CK-MB), myoglobin, and troponin, all of which assist in making the diagnosis of myocardial infarction, have proven unreliable for detecting acute myocardial ischemia 1, 2, 3. To be of any clinical value, currently available myocardial infarction markers must be repeated serially, at least 2 to 6 h after the onset of symptoms, to increase the likelihood of signaling an earlier ischemic event 4, 5. Consequently, the minimum hospital observation time required to rule out myocardial ischemia is usually 12 to 24 h and occasionally takes up to 3 days. Other tests may include serial EKGs, continuous cardiac monitoring (sometimes including real-time ST segment evaluations), and more definitive tests such as exercise treadmill, stress echocardiogram, radionucleotide stress imaging, or coronary angiography 6, 7. Numerous reports indicate that half or more of chest pain patients hospitalized for cardiac testing do not have any evidence of acute myocardial ischemia or acute coronary syndromes 1, 4. A rapid blood test that could rule out the presence of acute myocardial ischemia would dramatically improve the triage process of patients with acute coronary symptoms, eliminate many prolonged patient observation times, and reduce health care costs.

Although transitional metal binding to albumin is well known, alterations of human serum albumin (HSA) metal binding sites by ischemic events have not been extensively reported. HSA is a peptide consisting of 585 amino acids (66,500 Da) with a unique amino acid sequence, which may be specific to humans, at its amino terminus (“N-terminus”) (8). Previous studies have shown the N-terminus of HSA to be the primary binding site for the transitional metals Co(II), Cu(II), and Ni(II) (9). The HSA metal binding site is also particularly susceptible to biochemical degradation compared with albumin from other species (10).

While specifically looking for biochemical diagnostic markers from emergency patients’ serum, one author (D.B–O.) initially observed reduced in vitro binding of exogenous cobalt [Co(II)] to human serum albumin (HSA) in serum from several patients with acute coronary syndrome (unstable angina or very early myocardial infarction) (11). Based on that early observation, we postulated that significant molecular changes to or loss of portions of the N-terminus of HSA could reduce the in vivo transitional metal binding capacity of HSA either during myocardial ischemia or during reperfusion immediately after the ischemic event.

Prior reports indicate that the mechanisms involved in ischemia/reperfusion-induced in vivo changes to albumin may include exposure to endothelial and extracellular hypoxia, acidosis, free radical damage, membrane energy-dependent sodium and calcium pump disruptions, and free iron and copper ion exposure 12, 13, 14. Conditions necessary for altering the metal binding site of HSA are known to occur in vivo and probably occur within minutes after the onset of myocardial ischemia 13, 15. An assay based on changes in circulating HSA might demonstrate abnormal results minutes or hours before abnormal serum levels of myocardial infarction markers can be detected.

In this preliminary report, we describe a rapid colorimetric assay method measuring ischemia-induced alterations of the binding capacity of HSA to exogenous cobalt. Additionally, we report the results of a preliminary study testing the new assay on serum samples from emergency patients complaining of chest pain, including patients experiencing myocardial ischemia and acute coronary syndromes.