Myocardial ischemia, which precedes acute myocardial infarction (AMI)1 is associated with changes in human serum albumin (HSA) that result in decreased divalent cobalt ion (Co2+) binding. This reaction the basis of the serum colorimetric Co2+-HSA binding assay, which is an indirect measure of ischemia-modified albumin (IMA). Previously we showed that IMA was a useful diagnostic test for the diagnosis of myocardial ischemia in suspected acute coronary syndromes (ACS) patients (area under the ROC curve 0.95) (1). The IMA values are reversible between ischemic and nonischemic conditions (2). Furthermore, it is known that HSA is the primary binder of fatty acids, commonly known as free fatty acids (FFA), and that plasma concentrations of FFAs are increased during myocardial ischemia owing to a compensatory hyperadrenergic state. Considering these findings, we explored the hypothesis that FFA-induced conformational perturbations of HSA are the basis of the IMA test, and FFAs may themselves serve as a potential marker(s) of myocardial injury. Using pooled serum specimens, we performed an in vitro study to test the effect of physiologically relevant fatty acids on the IMA test. We obtained sera from patients brought to the emergency department at Kaiser Foundation Hospital who initially underwent a diagnostic work-up for ACS. …