Alterations in Unbound Free Fatty Acid Profiles in CVD

Project Summary- Patients with stable cardiovascular disease (CVD) have an elevated risk of a second event. The easiest method for screening risk in stable CVD patients would use biomarkers. However, no single marker has sufficient prognostic power. In this project we will determine whether profiles of unbound free fatty acids (FFAu) can help predict the risk of adverse outcomes in patients with stable cardiovascular disease (CVD). Plasma free fatty acids (FFA) which are mostly bound to albumin have long been associated with cardiac ischemia. Although present at only nM concentrations, the FFAu are the physiologically relevant fraction because the unbound and not the bound are transported into cells. We developed fluorescent probes (ADIFAB) to measure FFAu and found that FFAu levels were associated with cardiovascular ischemia during PTCA and in patients with STEMI in TIMI II. Human plasma is composed of about 30 unique FFA. Because they have distinct biological effects, we have developed a method to determine the different FFAu (profiles). The NHLBI provided us with plasma from 1200 patients (w/ clinical data) from the PEACE trial which sought to determine whether ACE inhibitors would improve outcomes in patients who had a cardiovascular event. We used our FFAu profiling method to profile 200 of the PEACE patients. For most of the FFAu there were no significant case (event) vs. control (no event) differences. However, for unbound alpha linolenate (αLNAu) the average case mole fractions were more than twice controls (plt0.0002). ROC analysis of the αLNAu concentrations in the 200 FFAu profiles yielded a C- index of 0.702, which is superior to the current 5 best biomarkers combined. This result suggests that αLNAu might be a novel risk factor that reflects events orthogonal to the current best biomarkers of stable CVD. Unexpectedly, two of the weakest binding FFAus, palmitoleate (POAu) and linolenate (αLNAu) had “zero” unbound, although both were present bound to albumin. Importantly, we found that blocking dissociation of POAu and α-LNAu occurs in blood but does not occur in aqueous media composed of albumin and FFA. In our FFAu profiles of healthy blood samples and non-cardiac diseases we found no measurable POAu and α-LNAu. This implies that there are specific inhibitors of POA and α-LNA dissociation from albumin in most blood samples, revealing a hitherto unknown mechanism for regulating FFA metabolism and a potential therapeutic target. Thus, FFAu profiles reveal biological effects that are invisible to total FFA profiles. In this project we will complete the measurement and analysis of the remaining unanalyzed PEACE samples and begin the search for the inhibitor of release.