Usefulness of Serum Unbound Free Fatty Acid Levels to Predict Death Early in Patients With ST-Segment (from the Thrombolysis In Myocar Andrew H. Huber, PhDa, J. Patrick Kampf, PhDb Jesse Adams III, MDd, and A Circulating total free fatty acid (FFA) levels a (MI) and have been associated with an increas ation of serum unbound FFA (FFAu) levels w ST-segment elevation MI in the Thrombolysi Thrombolysis In Myocardial Infarction II trial h a entr erap itali . Wh the 95% terv . Wo wice lar den � 20 occurs primaril tissue lipolysis cardiac tissue. albumin, a sma FFAu levels in of total FFA t physiologic ch rapidly, within balloon angiop levels from p Infarction (TIM (STEMI) could poor outcomes sented within 4 hours of STEMI onset with an intravenous lus heparin.9 r therapy, the percutaneous egy in which erwent percu- investigators and outcomes ccess data set present study. rum samples plasminogen was collected nvestigators11 art, Lung and th, Bethesda, bset of these y the National tween patient aFFA Sciences porated, San Dieg California; dUnive http://dx.doi.org/10.1016/j.amjcard.2013.08.057 information and blood specimen tube identification was maintained separately by the Maryland Medical Research Institute (Baltimore, Maryland). After our results had been deposited with the National Heart, Lung and Blood Institute, the linkage was unblinded. Complete patient information from the limited access data set was available for 1,834 patients of the 2,500 baseline blood specimens. Thus, we have reported the results for these 1,834 patients. The Institute, Louisville, Kentucky; and eTorrey Pines Institute for Molecular Studies, San Diego, California. Manuscript received April 28, 2013; revised manuscript received and accepted August 28, 2013. This work was supported by in part by grants DK070314 and DK058762 from the National Institutes of Health and in part by FFA Sciences LLC. See page 283 for disclosure information. *Corresponding author: Tel: (858) 455-3724; fax: (858) 455-3792. E-mail address:
[email protected] (A.M. Kleinfeld). 0002-9149/13/$ - see front matter � 2014 Elsevier Inc. All rights reserved. www.ajconline.org 12 hours. The MI-associated FFA increase y through catecholamine activation of adipose rather than FFA release from the ischemic 4 Although most plasma FFA is bound to ll fraction (3.8 years of follow-up factors, the FFAu levels in the fourth versus factor for death from MI (hazard ratio 5.0, death (hazard ratio 2.4, 95% confidence in ratio 1.9, 95% confidence interval 1.2 to 3.1) 2 FFAu quartiles and had approximately t FFAu elevation is 1 of the earliest molecu ST-segment elevation MI and was indepen outcomes after ST-segment elevation MI. Cardiol 2014;113:279e284) Plasma free fatty acid (FFA) levels are elevated early after acute myocardial infarction (MI) and have correlated with Elevation Myocardial Infarction dial Infarction [TIMI] II Trial) , Thomas Kwan, PhDb, Baolong Zhu, PhDc, lan M. Kleinfeld, PhDa,e,* re elevated early in myocardial infarction e in mortality. We investigated the associ- ith mortality in patients presenting with s In Myocardial Infarction II trial. The enrolled patients within 4 hours of chest recombinant tissue plasminogen activator ation was evaluated in serum samples from y. The FFAu level was an independent risk zation and continued to be an independent en adjusted for other cardiovascular risk first quartile remained an independent risk confidence interval 1.9 to 13.0), all cardiac al 1.3 to 4.4), and all-cause death (hazard men were twice as likely to be in the upper the rate of death as men. In conclusion, biomarkers of mortality in patients with t of other risk factors known to affect the 14 Elsevier Inc. All rights reserved. (Am J Methods udy p ) ) ardio Table 1 Baseline parameters for Thrombolysis In Myocardial Infarction (TIMI) II st Q1 (n ¼ 458) Q2 (n ¼ 458 FFAu (nmol/L) 1.9 (0.09e2.6) 3.2 (2.6e3.9 Age (yrs) 53.9 53.8 280 The American Journal of C present study complied with the Declaration of Helsinki, and the institutional review board committee of the Torrey Pines Institute for Molecular Studies approved the study. Measurements of the serum FFAu concentrations were performed using the fluorescent probe ADIFAB2 (FFA Sciences LLC, San Diego, California), as described previ- ously for patients with cardiac ischemia and MI8,12; however, Men 398 (87) 389 (85) White 398 (87) 398 (87) BMI (kg/m2) 27 (15e40) 27 (18e46) DBP (mm Hg) 81 (42e130) 81 (50e110) SBP (mm Hg) 130 (56e180) 130 (60e183) Creatine kinase (IU/L) 105 (5e1,760) 104 (12e3,590) Diabetes mellitus 41 (9) 37 (8) Previous MI 60 (13) 64 (14) Previous hypertension 174 (38) 156 (34) b Blocker within 24 h 87 (19) 87 (19) Data for ordinal parameters are presented as n (%), p values calculated using the (interquartile range), with p values calculated using the Kruskal-Wallis test. Ages were grouped to protect confidentiality and were proportional to the grou The baseline creatine kinase levels were elevated to more than the upper limit The mean interval from chest pain symptom onset to treatment initiation was v The presented demographic and clinical parameters were representative of the BMI ¼ body mass index; DBP ¼ diastolic blood pressure; Q ¼ quartile; SBP Table 2 Death and outcomes according to baseline unbound free fatty acid (FFAu) quarti Median FFAu (n Q1 (1.9) Q2 (3.2) Q Death from MI At 1 day 3 7 At 7 days 3 7 At 30 days 4 10 At 3.8 yrs 6 14 All cardiac causes At 1 day 3 7 At 7 days 3 7 At 30 days 5 11 At 3.8 yrs 16 27 All causes At 1 day 3 8 At 7 days 3 9 At 30 days 5 13 At 3.8 yrs 29 38 Noncardiovascular causes at 3.8 yrs* 13 9 Other outcomes Peak (8 h) creatine kinase (IU/L) 1,628 1,957 Data are presented as numbers of patients, with percentages in parentheses of tot all-cause cardiac plus noncardiac plus hemorrhage. p Values were determined using the chi-square test for all but peak creatine calculated using the Cochran-Armitage trend test. Q ¼ quartile. * An additional 12 patients died of hemorrhage during the 3.8 -year follow-up pe death without cause equaled 187. atients as a function of unbound free fatty acid (FFAu) quartile Q3 (n ¼ 459) Q4 (n ¼ 459) p Value 4.9 (3.9e6.4) 10.2 (6.46e523) 55.3 55.4 0.008 logy (www.ajconline.org) it was modified for 96-well plate fluorometry using a Flurolog 3 spectrofluorometer with a MicroMax plate reader (Horiba Ltd., Kyoto, Japan). Serum samples were diluted to 1% (vol/vol) in 200 ml of measuring buffer13 in 96-well plates. Fluorescence was measured after adding ADIFAB2 (1.5 mmol/L), and the intensities were used to determine the ratio (R) of the fluorescence intensities at 550 to 457 nm, 344 (75) 372 (81) FFAu. Survival curves for quartile (Q)1 to Q4 for death from (A)MI, (B) all cardiac causes, and (C) all causes. Log-rank probabilities were Variable HR 95% CI p Value ardio MI deaths Unadjusted Q2 2.3 0.9e5.9 0.094 Q3 4 1.6e9.8 0.002 Q4 5.6 2.3e13 10% of the 1,834 patients, and previous observed associations with FFAs. contribute to sudden cardiac death and death from MI.1e4 Moreover, these studies found that early death after MI correlated highly with the increasing total FFA levels.1 The total FFA levels also correlated with the long-term (6.9 years) increased risk of sudden cardiac death in the LUdwigshafen RIsk and Cardiovascular Health LURIC) study.15 Our results are consistent with, and additive to, the findings from the LURIC study.15 Both the present and LURIC studies found strong baseline correlations between the FFAu or total FFA levels and female gender or a history of diabetes (Table 1). The hazard ratios for MI deaths in the TIMI II study were larger (threefold or greater) and less sensitive to other risk factors than in the LURIC study. Presumably, this increase in sensitivity was a reflection of the acute presentation of STEMI in the TIMI II study and the high degree of FFAu sensitivity to cardiac ischemia. 7,8 The lower survival of women and those with diabetes compared with nondiabetic men (Table 3) is consistent with the findings from other studies of patients with STEMI.16,17 In the TIMI II study, the women had greater FFAu levels than did the men. The median FFAu for nondiabetic men and women was 3.7 and 4.5 nmol/L, respectively. The greater FFAu levels for women in the TIMI II study might reflect a gender-specific difference in the response to STEMI or might be a related to the older (7 years) age of the women in the TIMI II study, given that the FFAu levels increase with age (Table 1). However, the adjusted hazard ratio (Table 4) for death from MI for women in quartile 4 versus quartile 1 was 9.7 (95% confidence interval 0.971 to 100; p ¼ 0.053). These results and the higher death rates in quartiles 3 and 4 for women, raise the possibility that the FFAu level might be a more potent independent risk factor for women than for men. Although these results have demonstrated that FFAu levels correlate with early death after STEMI, causation is unproved. The increase in circulating FFAu concentrations might simply reflect the ischemia-induced activation of adipose lipolysis in proportion to the degree of ischemia and is probably largely generated from adipose tissue lipolysis stimulated by an ischemia-mediated increase in catechol- amine levels.3,4 In contrast, evidence for FFAs having a causal role has come from studies of nonischemic animals in which increasing circulating FFAs adversely affected myocardial metabolism, stimulated insulin resistance, induced arrhythmias, and increased cardiac enzyme release.18e21 Consistent with a causal role for FFAu in the TIMI II study was the increase in peak (8 hours), but not baseline, creatine kinase levels with increasing baseline FFAu level (Table 3). If FFAs adversely affect myocardial function, reducing the FFAu levels early after the ischemic event might reduce deaths and/or arrhythmias after MI.3,4 Infusions of glucose- insulin-potassium, shown to be protective in the dog,22 have been used to treat patients with STEMI and reduced circu- lating total FFA acutely in patients with STEMI.23 Although the effect on outcome in patients with STEMI has been mixed, it has been suggested that glucose-insulin-potassium treatment would be most effective if given at the earliest possible point.24,25 This concept was implemented in the logy (www.ajconline.org) Immediate Myocardial Metabolic Enhancement During Initial Assessment and Treatment in Emergency Care present with the greatest levels of FFAu. However, to confirm this, additional studies are necessary. tality Evidence-based guidelines currently recommend the rapid application of reperfusion therapy, with primacy given to percutaneous coronary artery intervention and limited use of a tissue plasminogen activator, the therapy used in the TIMI II trial. Nevertheless, the 30-day death rate in the TIMI II trial (4.2%) was not significantly different from the 5.4% rate reported for contemporary patients with STEMI.16 Adjustments for confounders that might affect the hazard ratios for FFAu were limited because important prognostic factors,27 including Killip class, baseline heart rate, number of diseased vessels, ejection fraction, smoking, and troponin level, were not available or were not recorded in the limited access data set. Angiotensin-converting enzyme inhibitors and statins were first approved for use in 1981 and 1987, respectively. TIMI II enrollment was between 1986 and 1988; therefore, most of these patients were unlikely to have been treated with these medications. Catecholamines have been associated, weakly, with mortality in patients with STEMI28 but were not measured. The beneficial effects of glucose-insulin-potassium suggest that FFAs, not catechol- amines, are important contributors to mortality in those with STEMI. The TIMI II protocols did not anticipate the determination of FFA levels; therefore, the blood samples might have had greater ex vivo than in vivo FFA levels because of lipoprotein lipolysis.29 However, this ex vivo effect would have obscured, rather than enhanced, the observed FFAu correlations with outcome. The determina- tion of FFAu concentrations requires knowledge of the relative distribution of the different FFAu present in serum and was estimated from the distribution of total FFAs, as described in previous studies.6,13 Differences in the FFAu distributions, such as those that can occur in acute coronary syndromes,30 are not expected to significantly alter the total FFAu levels. 6 The study was borderline underpowered for female patients, suggesting the need for additional studies. Acknowledgment: We thank the TIMI investigators, the National Heart, Lung and Blood Institute, and the Maryland Medical Research Institute. Disclosures Dr. Kleinfeld is the founder of, and major stock holder in, FFA Sciences LLC. Drs. Huber, Kampf, Kwan, and Zhu either are (A.H.H.) or were employees, are inventors on patents and applications assigned to FFA Sciences LLC, and (IMMEDIATE) trial by treating patients with STEMI with glucose-insulin-potassium in prehospital emergency medical settings.25,26 The IMMEDIATE trial revealed that glucose-insulin-potassium significantly reduced the FFA levels, reduced the incidence of cardiac arrest plus in- hospital mortality, and reduced the infarct size compared with patients not treated with glucose-insulin-potassium. Conceivably, the therapeutic efficacy of such interventions, using glucose-insulin-potassium or inhibitors of lipolysis, would be most evident in those patients with MI who Coronary Artery Disease/Mor have profit interests in FFA Sciences LLC. Dr. Adams is an advisor to FFA Sciences LLC. 1. Oliver MF, Kurien VA, Greenwood TW. Relation between serum-free- fatty acids and arrhythmias and death after acute myocardial infarction. Lancet 1968;291:710e714. 2. 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Am Heart J 2011;162:276e282. 284 The American Journal of Cardiology (www.ajconline.org) Usefulness of Serum Unbound Free Fatty Acid Levels to Predict Death Early in Patients With ST-Segment Elevation Myocardial ... Methods Results Discussion Acknowledgment Disclosures References