Utility of Serum Fatty Acid Concentrations as a Marker for Acute Myocardial Infarction and Their Potential Role in the Formation of Ischemia-Modified Albumin: A Pilot Study

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. Our pilot study research protocol was approved by the Institutional Review Board of Kaiser Foundation Hospital (Honolulu, HI). All serum samples came from the same pool of patients with and without AMI on the basis of final discharge diagnosis. The study was double-blinded. The clinical assessment of AMI included several objective clinical indices, including imaging studies, electrocardiogram studies, serum cardiac biochemical markers, and the criteria defined by the Joint European Society of Cardiology/American College of Cardiology Committee. Patients with renal disease, thyroid disease, or diabetes mellitus; with a body mass index >35; or with serum albumin of <30 g/L and >55 g/L were excluded from this study. The AMI group comprised 15 males and 19 females, and their mean age was 64.5 years. The non-AMI group consisted of 10 males and 14 females with a mean age of 62.8 years. We measured serum FFA concentrations by using a colorimetric nonesterified fatty acid determination kit (Wako Chemicals) and measured IMA values by using reagents and standards provided by Inverness Medical. We also quantified specific serum FFA concentrations in non-AMI and AMI individuals (n = 13 per group) by using organic solvent extraction, thin-layer chromatography, transesterification, and capillary-zone electrophoresis (3). All values are expressed as mean ±2 SD. Statistical significance was assessed by Wilcoxon tests and P values <0.05 were considered significant. In the in vitro studies performed using pooled sera, addition of fatty acids showed elevations in IMA values. Oleate and arachidonate additions at a comparable fatty acid/HSA molar ratio of about 8 produced a significant increase in the percentage IMA values by 32.3 ± 0.8 and 37.9 ± 0.5, respectively. Serum FFA concentrations of persons with and without AMI were 1.03 ± 0.45 mmol/L and 0.77 ± 0.34 mmol/L, respectively (P < 0.018), and in the same 2 groups the IMA values were 119.4 ± 37.3 U/mL and 88.6 ± 19.3 U/mL, respectively (P < 0.0005). The ratios of mean values of FFA (1.34) and IMA (1.35) for AMI and non-AMI individuals are virtually identical, suggesting a temporal and proportional relationship. The correlation coefficient between FFA and IMA values in our test patients was 0.3293 (P = 0.012), indicating a statistically significant positive relationship. In the analysis of individual FFAs, 5 fatty acids, oleic, palmitic, linoleic, stearic, and palmitoleic acid, showed statistically significant elevations in AMI patients compared to non-AMI individuals (Table 1 ). Several potential cardiac markers for the diagnosis of ACS syndrome continue to be investigated (4). Our study shows that total FFA concentrations as well as specific FFAs are increased in AMI and may have potential diagnostic value. Changes in IMA values during AMI are likely caused by reversible conformational changes in HSA that are associated with FFA fluxes. The formation of IMA during ACS has been attributed to the modifications that may occur in N-terminal region of albumin, because Co2+ is thought to bind primarily to this site. However, a recent study has shown that the Co2+ binding site(s) are not located at the N-terminus (5). Because N-terminal sequence analysis of purified HSA obtained from ischemic patients with high IMA values did not show N-terminal modifications (1), other sites are likely involved, leading to perturbations in the conformation of albumin. Our study suggests a plausible but not a causal relationship between FFA and IMA, and a potential role for measurement of total FFA and specific FFAs in ACS. Quantitative serum concentrations for 6 FFA levels.1 Values are mean ±2 SD, and showed significant increases (P < 0.05) except for arachidonic acid in the AMI compared to non-AMI patients. Each group consisted of 13 serum samples. Statistically not significant. Quantitative serum concentrations for 6 FFA levels.1 Values are mean ±2 SD, and showed significant increases (P < 0.05) except for arachidonic acid in the AMI compared to non-AMI patients. Each group consisted of 13 serum samples. Statistically not significant. Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures of Potential Conflicts of Interest:Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. Editor’s Note: Inverness Medical Innovations has notified their customers that effective June 30, 2009 that they will discontinue the sale and distribution of the Albumin Cobalt Binding Test due to insufficient demand to sustain production. Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript. Acknowledgments: We thank Hollie Huff of Inverness Medical Innovations for providing the commercial IMA test kits for our study.