Clinica Chimica Acta 438 (2015) 255–260
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Prognostic relevance of ischemia-modified albumin and NT-proBNP in patients with peripheral arterial occlusive disease Jürgen Falkensammer a,b,⁎, Andreas Frech b, Nikolaus Duschek a, Simon Gasteiger b, Tatjana Stojakovic c, Hubert Scharnagl c, Kurt Huber d, Gustav Fraedrich b, Andreas Greiner b,e a
Department of Vascular and Endovascular Surgery, Wilhelminen hospital, Vienna, Austria Department of Vascular Surgery, Medical University Innsbruck, Austria Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria d 3rd Medical Department, Cardiology, Wilhelminen hospital, Vienna, Austria e Department of Vascular Surgery, University Hospital Aachen, Germany b c
a r t i c l e
i n f o
Article history: Received 25 July 2014 Received in revised form 25 August 2014 Accepted 25 August 2014 Available online 3 September 2014 Keywords: Peripheral arterial disease Cardiovascular risk Oxidative stress Ischemia-modified albumin High-sensitivity cardiac Troponin T NT-proBNP
a b s t r a c t Background: Cardiovascular morbidity is high among patients with peripheral arterial occlusive disease (PAOD). The aim of this study was to evaluate the ability of ischemia-modified albumin (IMA), N-terminal proBNP (NTproBNP), and high-sensitive cardiac Troponin T (hs-cTnT) to predict cardiovascular complications in male patients with Fontaine stage II PAOD. Methods: 68 men with stage II PAOD underwent treadmill testing. NT-proBNP, IMA and hs-cTnT were measured before and after exercise. Patients were followed up prospectively and complete follow-up data were available for 66 individuals. Results: Median follow-up time was 43.0 months. 12 (18.2%) patients had suffered from a major adverse cardiac event (MACE). IMA and NT-proBNP baseline concentrations were significantly higher in patients who developed MACE during follow-up: IMA: 110.6 ± 2.4 kU/L vs. 102.5 ± 0.9 kU/L (p b 0.001); NT-proBNP: 270.5 ± 295.9 ng/L vs. 84.6 ± 15.4 ng/L (p = 0.007). In multivariable regression models only IMA was significantly associated with the primary endpoint (HR = 1.07, CI 1.01–1.13; p = 0.029). Conclusion: In the present study, a serum concentration of N 103.9 kU/L of IMA was a better independent predictor of MACE than NT-proBNP or hs-cTnT. IMA might be a valuable tool for risk stratification in PAOD patients. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Atherosclerosis is the main cause of cardiovascular morbidity and mortality worldwide. Significant affections of multiple arterial beds will be found in 34% of patients with atherosclerotic disease [1]. Peripheral arterial occlusive disease (PAOD) affects 15 to 20% of the elderly population in the industrialized countries [2]. PAOD patients suffer from annual rates of heart attack, stroke, and hospitalization that are comparable to or greater than rates observed in patients with
Abbreviations: PAOD, peripheral arterial occlusive disease; CAD, coronary artery disease; BNP, B-type natriuretic peptide; NT-proBNP, n-terminal pro-B type natriuretic peptide; cTn, cardiac Troponin; IMA, Ischemia-modified albumin; ACB-Test, albumin cobalt binding test; ROS, reactive oxygen species; MACE, major adverse cardiovascular event; AMI, acute myocardial infarction; CV, coefficient of variation; BCG, bromocresol green; IQR, interquartile range; BMI, body mass index; CI, confidence interval. ⁎ Corresponding author at: Department of Vascular and Endovascular Surgery, Wilhelminen hospital, Montleartstrasse 37, 1160 Vienna, Austria. Tel.: + 43 149150 4101; fax: +43 149150 4109. E-mail address: [email protected] (J. Falkensammer).
http://dx.doi.org/10.1016/j.cca.2014.08.031 0009-8981/© 2014 Elsevier B.V. All rights reserved.
established coronary artery disease (CAD) [3,4]. Accordingly, their prognosis is characterized by the degree of their cardiovascular comorbidity: Patients with Fontaine stage I or II PAOD have a 2% to 3% annual incidence of non-fatal myocardial infarction and total mortality is two to three times higher than that of an age-matched population [2,5]. Determination of a serum biomarker that is associated with coronary artery disease could help to identify PAOD patients who are at increased risk of cardiovascular complications and thus improve risk assessment, treatment and prognosis. Several serum markers including natriuretic peptides, troponins and ischemia-modified albumin have previously been proposed as prognostic markers in patients with heart disease. B-type natriuretic peptide (BNP) is synthesized in ventricular myocardium and it is released into the circulation in response to ventricular dilatation, pressure overload or myocardial ischemia [6,7]. Serum concentrations of natriuretic peptides including BNP or the inactive precursor NT-proBNP are increased in patients with cardiac disease, particularly in those with heart failure [7]. Recent studies have identified BNP as an important indicator of prognosis in patients with heart failure and myocardial infarction: elevated concentrations
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identify patients at high risk of progressive ventricular dilatation, heart failure, or death [8,9]. The release of cardiac Troponins (cTn) into the circulation is a consequence of myocardial injury. Measurement of cTn serum concentrations is a key diagnostic tool for detection of myocardial infarction [10]. New high-sensitivity cardiac Troponin (hs-cTnT) assays allow detection of low-level increases of cTn that are not necessarily associated with myocardial ischemia related to coronary hypoperfusion but rather with a more chronic, structural tissue damage. Recent investigations have revealed an association of higher baseline concentrations of cardiac Troponins with cardiovascular risk in asymptomatic individuals [11–13]. Ischemia-modified albumin (IMA) has been initially proposed as a marker for the diagnosis of myocardial ischemia [14,15]. Metabolic changes associated with ischemia result in a biochemical alteration of the N-terminus of albumin, thereby reducing its affinity to transition metals like cobalt. The albumin cobalt binding (ACB) test has been developed to quantify ischemia modified albumin (IMA) in serum by measuring the concentration of unbound cobalt after adding a defined quantity of cobalt to the serum sample [16,17]. However, IMA is not a tissue-specific marker of ischemia. Several authors have suggested that the generation of IMA from serum albumin is the consequence of contact with reactive oxygen species (ROS) [18,19]. Elevated baseline concentrations have been found in individuals with excessive chronic oxidative stress levels, including patients with end-stage renal disease [20], morbid obesity [19], diabetes mellitus [21] or hypercholesterolemia [18] and affected patients appear to be at an increased risk for cardiovascular complications [22–24]. This study aimed to assess the prognostic value of IMA, BNP and hscTNT for long-term occurrence of major adverse cardiovascular events (MACE) and mortality in ambulatory patients with Fontaine stage II peripheral arterial disease. 2. Methods This investigation was designed as a prospective observational study and patients were recruited in the outpatient clinic of the department of vascular surgery of the Medical University Innsbruck. Because of a lack of preliminary data on the predictive value of IMA on the occurrence of MACE in a comparable patient collective, a power calculation could not be performed. This was a hypothesis-generating investigation. 2.1. Patients Sixty-eight men with stable Fontaine stage II PAD participated in this study. The study protocol has been described previously [25,26]. All patients underwent constant-load treadmill testing at 3 km/h and 12% inclination, to maximum walking distance. Patients with symptomatic CAD, acute myocardial infarction (AMI) within the previous three months and exercise limiting orthopedic or respiratory diseases as well as patients receiving digoxin were not included. All patients included had a normal or only mildly reduced renal function with a glomerular filtration rate of N50 mL/min/1.73 m 2 as calculated by the MDRD-formula (Modification of Diet in Renal Disease; 186 × (Creat / 88.4) − 1.154 × (Age) − 0.203). Body mass index (BMI) was calculated as weight (kg) / height (m)2. During annual visits in the outpatient clinic, participants were monitored for the occurrence of major adverse cardiovascular events (fatal and non-fatal myocardial infarction, coronary artery intervention, coronary artery bypass graft, stroke). Patients who missed their appointment were contacted via telephone and a separate appointment was scheduled. Data of deceased patients were crosschecked with the hospitals' patient documentation system. The study protocol was approved by the institutional research ethics committee and all participants gave written informed consent for participation in the study. Performance of the study complied with the
World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects and/or animals. 2.2. Sample acquisition and handling Before the start of exercise, a short 18-gauge venous catheter (Venflon™, Viggo-Spectramed, Helsingborg, Sweden) was inserted into either the antecubital cephalic vein or the cubital vein of the right arm. Blood was directly drawn into serum and lactate tubes, respectively (S-Monovette®, Sarstedt AG & Co., Nuremberg, Germany) before exercise (=baseline) and 5, 10 and 30 min after cessation of treadmill testing, respectively. The tubes were immediately stored on crushed ice and processed within 60 min by centrifugation at 2500 ×g for 15 min and stored of 500 μL aliquots at − 80 °C. The samples were shipped to the Clinical Institute of Medical and Chemical Laboratory Diagnostics at the Medical University of Graz. N-terminal pro-B natriuretic peptide (NT-proBNP) concentrations were determined with an Elecsys 2010 instrument (Roche Diagnostics, Mannheim, Germany), with a lower detection limit of 5 ng/L and a cutoff value of 125 ng/L. According to the manufacturer the interassay coefficient of variation (CV) was 4.5% for 200 ng/L and 3.6% for 4002 ng/L of quality control material and we found CVs of 3.10% and 2.05% for low and high quality control material, respectively. Serum IMA was measured using the albumin cobalt binding test (Ischemia Technologies Inc., Denver, CO) on a Roche Modular P instrument (Roche Diagnostics, Basel, Switzerland). According to the manufacturer, normal values determined in a population of 283 healthy individuals ranged from 52 to 116 kU/L, resulting in a cut-off value of 85 kU/L (95th percentile). The total interassay CV was 2.8% for quality-control material (mean of three control materials with different concentrations) as determined in the laboratory (Medical University of Graz). Due to the previously described interdependency of IMA and serum albumin, calculation of an IMA/albumin coefficient (kU/g) was performed. Serum albumin was measured with the BCG (bromocresol green) method using enzymatic reagents and standards from Roche. According to the manufacturer the interassay CV for albumin measurements was 1.7%. High sensitive Troponin T (hsTnT) was measured on an Elecsys system (Roche Diagnostics, Mannheim, Germany) using the Troponin T hs test and standards from Roche. The manufacturer reported the interassay CV as 2.6% for 27.9 ng/L and 1.7% for 2049 ng/L of quality control material and we found CVs of 2.96% and 2.12% for low and high quality control material, respectively. According to the manufacturer, the limit of blank is 3 ng/L and 5 ng/L is given as detection limit. The upper reference value was set at 14 ng/L, reflecting the 99th percentile of normal values determined in a population of 546 healthy individuals. 2.3. Statistics The skewed statistical distribution of baseline biomarker concentrations at baseline prompted non-parametric tests (Mann–Whitney-U) for comparison of medians. Quantitative changes of serum concentrations of biomarkers after treadmill testing were analyzed as raw and percentile change scores. Kaplan–Meier-curves (log-rank test) were used for survival analysis of halves (percentiles) of biomarker concentrations. In order to adjust for potential confounders, multivariable proportional hazard models (Cox Regression) including major cardiovascular risk factors (CVRF; age, body mass index (BMI), smoking, diabetes, hypertension, hypercholesterolemia, history of cardiovascular disease) were used to estimate hazard ratios (HR; 95% confidence interval (CI)) for the occurrence of the primary endpoint. The primary combined endpoint comprised major adverse cardiovascular events. Pairwise comparison of significant biomarkers (NT-pro BNP and IMA) was performed in uniand multivariate proportional hazard models using backward elimination removing non-significant variables with MACE as primary endpoint. A two-sided p-value (P) of less than 0.05 was considered to indicate
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statistical significance. All analyses were performed with the use of SPSS 20 (IBM Inc., Somers, NY 10589, USA.).
Two patients were lost to follow-up and excluded from the analysis. Characteristics and distribution of baseline cardiovascular markers of the 66 patients with complete follow-up are presented in Table 1. The observation period lasted from the date of first sample retrieval until the date of last follow-up (median observation period 43.0 months) for all 66 male patients. After the observation period 12 (18.2%) of 66 patients had suffered from a cardiovascular adverse event (primary endpoint; Table 2). Comparison of medians of selected biomarkers showed significantly different serum concentrations of IMA, the ratio of IMA/Albumin and NT-pro BNP in patients with MACE compared to event-free individuals (Table 3). Noteworthy, in multivariable regression models (Cox Regression) correcting for confounders (age, BMI, smoking, diabetes, hypertension, hypercholesterolemia, history of cardiovascular disease) only IMA was significantly (HR = 1.07, CI 1.01– 1.13; p = 0.029) associated with the primary combined endpoint as shown in Table 4. Post-exercise concentrations and quantitative changes of serum concentrations of biomarkers (raw and percentile change scores) after treadmill testing showed no significant association with
Table 1 Characteristics and distribution of baseline cardiovascular markers of patients (n = 68) with peripheral occlusive disease. Dichotomous
Count
Percent (%)
Risk factors Smoking Diabetes Hypertension Hypercholesterolemia Coronary artery disease Cerebrovascular disease
65 9 40 51 20 9
95.6% 13.2% 58.8% 76.1% 29.4% 13.2%
Medication ASA Clopidogrel Heparin Coumadin β-Blocker ACE-inhibitor
55 12 1 6 15 29
80.9% 17.6% 1.5% 8.8% 22.1% 42.6%
Continuous
Median
IQR
Age (years) BMI Maximum walking distance (meter) IMA (kU/L) Albumin (g/L) IMA/Albumin (kU/g) Hs-cTnT (ng/L) NT-pro BNP (ng/L)
66.0 23.0 185.0 103.7 4.4 2.3 6.7 84.3
10.9 5.0 175.0 10.0 0.4 0.5 5.7 92.5
ASA acetylsalicylic acid, BMI body mass index, hs-cTnT high sensitivity cardiac Troponin T, IMA ischemia modified albumin, IQR interquartile range, MCI myocardial infarction, NT-pro BNP N-terminal pro-B natriuretic peptide.
Table 2 Adverse events.
Cardiovascular event (any) Cerebrovascular event AMI Deceased Stroke, fatal AMI, fatal AMI denotes acute myocardial infarction.
Table 3 Comparison of medians of cardiovascular markers by occurrence of primary endpoint (MACE) of 68 patients with peripheral arterial occlusive disease. p-Value by Mann– Whitney-U test. Marker
3. Results
Count
Percent (%)
12 3 9 5 1 4
18.2% 4.5% 13.6% 7.6% 1.5% 6.1%
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IMA (kU/L) IMA/Albumin (kU/g) hs-cTnT (ng/L) NT-pro BNP (ng/L)
Alive
MACE
p-Value
Median
±SE
Median
±SE
102.5 2.3 6.6 84.6
0.9 0.0 0.7 15.4
11.6 2.6 9.4 270.5
2.4 0.1 1.5 295.9
b0.001 0.001 0.356 0.007
hs-cTnT denotes high sensitivity cardiac Troponin T, IMA ischemia modified albumin, NT-pro BNP N-terminal pro-B natriuretic peptide and SE standard error.
occurrence of the primary endpoint (p N 0.05), as presented in Table 5. Kaplan–Meier analysis demonstrated a significantly higher event-free survival rate after 4 years (96.9% versus 66.7%) in patients with lower IMA baseline concentrations (Fig. 1) as well as a significantly decreased cardiac mortality (0% versus 12.1%; Supplemental Fig. 1). After adjusting for confounders, this correlated to an 11.3 time higher risk of suffering a MACE within the observation period (HR = 11.3, CI 1.3–93.1; p = 0.024; Supplemental Fig. 2). As prognostic marker, IMA showed a better discriminatory power (AUC 0.84 CI 0.70–0.97; PI b 0.001) than Nt-proBNP or hs-cTnT (Fig. 2).
Table 4 Uni- and multivariate analyses estimating the association of cardiovascular biomarkers of 66 patients with cardiovascular adverse events (primary endpoint) within the observation time. p-Value by proportional hazard model (Cox Regression). Multivariable model adjusted for cardiovascular risk factors (age, BMI, smoking, diabetes, hypertension, hypercholesterolemia, history of cardiovascular disease). HR
95.0% CI
p-Value
Lower
Upper
Univariate IMA IMA/Albumin hs-cTnT NT-pro BNP
1.08 4.05 1.05 1.00
1.03 1.41 0.94 1.00
1.13 11.62 1.17 1.00
0.003 0.009 0.394 0.024
Multivariate IMA IMA/Albumin hs-cTnT NT-pro BNP
1.07 3.13 1.04 1.00
1.01 0.95 0.92 1.00
1.13 10.35 1.16 1.00
0.029 0.061 0.562 0.061
CI denotes confidential interval, hs-cTnT high-sensitivity cardiac Troponin T, IMA ischemia modified albumin and NT-pro BNP N-terminal pro-B natriuretic peptide.
Table 5 Multivariable analysis of the effect of exercise-dependent serum changes of cardiovascular markers of 66 patients on MACE (primary endpoint) within the observation time. p-Value by proportional hazard model (Cox Regression). Multivariable model adjusted for cardiovascular risk factors (age, BMI, smoker, diabetes, hypertension, hypercholesterolemia, history of cardiovascular disease). HR
95.0% CI (HR)
p-Value
Lower
Upper
RCS multivariate IMA IMA/Albumin hs-cTnT NT-pro BNP
1.05 1.60 1.38 1.00
0.96 0.15 0.87 0.97
1.15 17.12 2.18 1.03
0.306 0.700 0.172 0.857
PCS multivariate IMA IMA/Albumin hs-cTnT NT-pro BNP
1.05 1.01 1.02 1.02
0.96 0.95 0.99 0.96
1.15 1.07 1.04 1.10
0.278 0.776 0.164 0.511
HR denotes hazard ratio, hs-cTnT high sensitivity cardiac Troponin T, IMA ischemia modified albumin, NT-pro BNP N-terminal pro-B natriuretic peptide.
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Fig. 1. Survival estimate (KM; Kaplan Meier curve) for MACE of patients (n = 66) with PAOD by IMA b median compared to IMA ≥ median. p-Value by log-rank test.
4. Discussion Our data provide strong evidence that in patients with Fontaine stage II PAOD, high concentrations of IMA are a strong and independent predictor of cardiovascular complications and overall mortality after an average follow-up interval of 3.6 years. A multivariable logistic regression analysis adjusted for classic risk factors including age, BMI, smoking status, diabetes, hypertension and history of cardiovascular disease identified IMA at baseline as the only independent predictor of reaching the primary end point.
This is in accordance with recent publications. An association of elevated serum concentrations of ischemia-modified albumin with shortterm mortality has been reported after acute myocardial infarction [27] and following cardiopulmonary resuscitation in cardiac arrest patients [28]. An increased risk of major cardiovascular complications as well as a higher 1-year mortality have been found in patients with increased baseline concentrations of IMA after admission for acute coronary syndrome [22,23] and after acute myocardial infarction [29]. Elevated IMA concentrations have been found in diseases that are associated with excessive chronic oxidative stress levels and also with an increased mortality, including end-stage renal disease [20], morbid obesity [19], diabetes mellitus [21] or hypercholesterolemia [18]. Oxidative stress plays a major role in the onset and progression of atherosclerosis [30]. Reactive oxygen species are produced by diseased endothelial cells as well as smooth muscle cells and increased shear stress can also stimulate superoxide production [30]. If the generation of IMA from serum albumin is the consequence of contact with reactive oxygen species, as has been suggested previously [18,19], IMA might in fact be a serum marker for the severity of systemic atherosclerosis. The potential value of IMA for the prediction of adverse cardiovascular events is supported by two important observations. It is independent from concentration changes induced by moderate exercise, as shown through the treadmill testing. A potential association of stress-induced changes of serum IMA concentrations with future adverse events has previously only been investigated once: Sharma et al. reported a significant association of dobutamine stress test induced increase of IMA ≥ 20 kU/L with cardiac as well as all-cause mortality at 1 year in patients with end-stage renal disease while IMA baseline concentrations were not associated with an increased risk [24]. However, the amount of (cardiac) stress during treadmill exercise is certainly not comparable to dobutamine stress testing, especially in a population of patients with a claudication-limited walking distance. Furthermore, the predictive value of IMA does not seem to be affected by concomitant albumin concentrations, as IMA/Alb ratios have not shown a significant association with the occurrence of the primary outcome. Although initial reports claimed that the ACB test would be
Fig. 2. ROC curves estimating discriminatory power (AUC; area under the curve) of selected biomarkers for occurrence of MACE (primary endpoint). Ideal marker AUC N 0.8. hs-cTnT denotes high sensitivity cardiac Troponin T, IMA ischemia modified albumin and NT-pro BNP N-terminal pro-B Natriuretic Peptide. AUC area under the ROC curve, CI confidential interval. C-Index corresponds to an AUC of 0.5 with no discriminatory power.
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insensitive to serum albumin concentrations [31], previous publications have highlighted a negative correlation of serum albumin and IMA [32–34]. Concerning the predictive value of IMA, this relationship appears to be insignificant. Preoperative NT-proBNP concentrations have also been shown to predict myocardial injury or peri- and postoperative death in patients undergoing major non-cardiac vascular surgery [35,36]. The predictive value of NT-proBNP in patients with peripheral arterial disease has also been investigated previously: Mueller et al. reported a significantly increased risk of death of all causes in patients with serum NT-proBNP concentrations above the median of 213 ng/L in a population of 418 patients with peripheral arterial disease including a proportion of 19% of cases with critical limb ischemia [37]. Although we did find a somewhat increased risk of cardiovascular complications in patients with NTproBNP concentrations above the median of 89.7 ng/L in our population, this was not significant in the multivariable analysis. In any case, comparison of ROC curves for IMA and NT-proBNP showed that IMA baseline concentrations were superior in predicting MACE, independent of the selected cutoff-value. Recent publications pointed to a potential value of cardiac Troponins as prognostic markers in addition to their value for the diagnosis of acute cardiac events: Otsuka et al. have reported an association of low-level increases of hs-cTnT in middle-aged men without overt CAD with the Framingham risk prediction score for coronary artery disease and suggested using hs-cTnT for identification of high-risk patients for the primary prevention of CAD [11]. Recently, it has been shown that elevated baseline concentrations of hs-cTnT are a predictor of cardiovascular mortality not only in patients with established coronary artery disease [12] but also in the general population [13]. However, in our collective of Fontaine stage IIb PAOD patients, we did not find an association of either baseline or post-exercise serum concentrations of hscTnT with cardiovascular complications. A limitation of the current investigation is the relatively low number of patients included. However, the low drop out rate of 2.9% during follow-up underlines the high quality of study performance. Moreover, our study only included male patients with unknown, symptom-free coronary or cerebral artery disease and results cannot be applied to female patients. 5. Conclusions In our population of male patients with Fontaine stage II PAOD with unknown, symptom-free coronary or cerebral artery disease, baseline serum concentration of IMA was a strong independent predictor of cardiovascular adverse events. Thus, IMA might serve as a valuable risk marker for future cardiovascular events and cardiovascular mortality. In addition, baseline IMA concentrations are independent from stressinduced changes and not affected by serum albumin concentrations. Our findings are hypothesis-generating and should be further investigated in a larger patient collective, including female individuals. The potential value of IMA as a serum marker for the severity of systemic atherosclerosis must be assessed in a collective that is well defined as far as coronary artery disease, carotid artery disease as well as PAOD are concerned. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2014.08.031. Acknowledgments The technical assistance of the laboratory staff at the Medical University of Graz is gratefully acknowledged. Roche Diagnostics provided assay reagent sets free of charge but did not assume any other role in the conduct of the study. We thank the Society of Atherosclerosis, Thrombosis and Vascular Biology, ATVB20090501 (Verein ATVB), Vienna, Austria, for financially supporting determinations of ischemia-modified albumin.
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[26] Falkensammer J, Gasteiger S, Stojakovic T, Stuhlinger M, Scharnagl H, Frech A, et al. Elevated baseline hs-cTnT levels predict exercise-induced myocardial ischemia in patients with peripheral arterial disease. Clin Chim Acta 2012;413:1678–82. [27] Dominguez-Rodriguez A, Abreu-Gonzalez P, Jimenez-Sosa A, Samimi-Fard S, Idaira HB. Does ischemia-modified albumin add prognostic value to the Thrombolysis In Myocardial Infarction risk score in patients with ST-segment elevation myocardial infarction treated with primary angioplasty? Biomarkers 2009;14:43–8. [28] Turedi S, Gunduz A, Mentese A, Dasdibi B, Karahan SC, Sahin A, et al. Investigation of the possibility of using ischemia-modified albumin as a novel and early prognostic marker in cardiac arrest patients after cardiopulmonary resuscitation. Resuscitation 2009;80:994–9. [29] Van Belle E, Dallongeville J, Vicaut E, Degrandsart A, Baulac C, Montalescot G. Ischemia-modified albumin levels predict long-term outcome in patients with acute myocardial infarction The French Nationwide OPERA study. Am Heart J 2010;159: 570–6. [30] Harrison D, Griendling KK, Landmesser U, Hornig B, Drexler H. Role of oxidative stress in atherosclerosis. Am J Cardiol 2003;91:7A–11A. [31] Apple FS, Wu AH, Mair J, Ravkilde J, Panteghini M, Tate J, et al. Future biomarkers for detection of ischemia and risk stratification in acute coronary syndrome. Clin Chem 2005;51:810–24.
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Prognostic relevance of ischemia-modified albumin and NT-proBNP in patients with peripheral arterial occlusive disease Jürgen Falkensammer a,b,⁎, Andreas Frech b, Nikolaus Duschek a, Simon Gasteiger b, Tatjana Stojakovic c, Hubert Scharnagl c, Kurt Huber d, Gustav Fraedrich b, Andreas Greiner b,e a
Department of Vascular and Endovascular Surgery, Wilhelminen hospital, Vienna, Austria Department of Vascular Surgery, Medical University Innsbruck, Austria Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria d 3rd Medical Department, Cardiology, Wilhelminen hospital, Vienna, Austria e Department of Vascular Surgery, University Hospital Aachen, Germany b c
a r t i c l e
i n f o
Article history: Received 25 July 2014 Received in revised form 25 August 2014 Accepted 25 August 2014 Available online 3 September 2014 Keywords: Peripheral arterial disease Cardiovascular risk Oxidative stress Ischemia-modified albumin High-sensitivity cardiac Troponin T NT-proBNP
a b s t r a c t Background: Cardiovascular morbidity is high among patients with peripheral arterial occlusive disease (PAOD). The aim of this study was to evaluate the ability of ischemia-modified albumin (IMA), N-terminal proBNP (NTproBNP), and high-sensitive cardiac Troponin T (hs-cTnT) to predict cardiovascular complications in male patients with Fontaine stage II PAOD. Methods: 68 men with stage II PAOD underwent treadmill testing. NT-proBNP, IMA and hs-cTnT were measured before and after exercise. Patients were followed up prospectively and complete follow-up data were available for 66 individuals. Results: Median follow-up time was 43.0 months. 12 (18.2%) patients had suffered from a major adverse cardiac event (MACE). IMA and NT-proBNP baseline concentrations were significantly higher in patients who developed MACE during follow-up: IMA: 110.6 ± 2.4 kU/L vs. 102.5 ± 0.9 kU/L (p b 0.001); NT-proBNP: 270.5 ± 295.9 ng/L vs. 84.6 ± 15.4 ng/L (p = 0.007). In multivariable regression models only IMA was significantly associated with the primary endpoint (HR = 1.07, CI 1.01–1.13; p = 0.029). Conclusion: In the present study, a serum concentration of N 103.9 kU/L of IMA was a better independent predictor of MACE than NT-proBNP or hs-cTnT. IMA might be a valuable tool for risk stratification in PAOD patients. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Atherosclerosis is the main cause of cardiovascular morbidity and mortality worldwide. Significant affections of multiple arterial beds will be found in 34% of patients with atherosclerotic disease [1]. Peripheral arterial occlusive disease (PAOD) affects 15 to 20% of the elderly population in the industrialized countries [2]. PAOD patients suffer from annual rates of heart attack, stroke, and hospitalization that are comparable to or greater than rates observed in patients with
Abbreviations: PAOD, peripheral arterial occlusive disease; CAD, coronary artery disease; BNP, B-type natriuretic peptide; NT-proBNP, n-terminal pro-B type natriuretic peptide; cTn, cardiac Troponin; IMA, Ischemia-modified albumin; ACB-Test, albumin cobalt binding test; ROS, reactive oxygen species; MACE, major adverse cardiovascular event; AMI, acute myocardial infarction; CV, coefficient of variation; BCG, bromocresol green; IQR, interquartile range; BMI, body mass index; CI, confidence interval. ⁎ Corresponding author at: Department of Vascular and Endovascular Surgery, Wilhelminen hospital, Montleartstrasse 37, 1160 Vienna, Austria. Tel.: + 43 149150 4101; fax: +43 149150 4109. E-mail address: [email protected] (J. Falkensammer).
http://dx.doi.org/10.1016/j.cca.2014.08.031 0009-8981/© 2014 Elsevier B.V. All rights reserved.
established coronary artery disease (CAD) [3,4]. Accordingly, their prognosis is characterized by the degree of their cardiovascular comorbidity: Patients with Fontaine stage I or II PAOD have a 2% to 3% annual incidence of non-fatal myocardial infarction and total mortality is two to three times higher than that of an age-matched population [2,5]. Determination of a serum biomarker that is associated with coronary artery disease could help to identify PAOD patients who are at increased risk of cardiovascular complications and thus improve risk assessment, treatment and prognosis. Several serum markers including natriuretic peptides, troponins and ischemia-modified albumin have previously been proposed as prognostic markers in patients with heart disease. B-type natriuretic peptide (BNP) is synthesized in ventricular myocardium and it is released into the circulation in response to ventricular dilatation, pressure overload or myocardial ischemia [6,7]. Serum concentrations of natriuretic peptides including BNP or the inactive precursor NT-proBNP are increased in patients with cardiac disease, particularly in those with heart failure [7]. Recent studies have identified BNP as an important indicator of prognosis in patients with heart failure and myocardial infarction: elevated concentrations
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identify patients at high risk of progressive ventricular dilatation, heart failure, or death [8,9]. The release of cardiac Troponins (cTn) into the circulation is a consequence of myocardial injury. Measurement of cTn serum concentrations is a key diagnostic tool for detection of myocardial infarction [10]. New high-sensitivity cardiac Troponin (hs-cTnT) assays allow detection of low-level increases of cTn that are not necessarily associated with myocardial ischemia related to coronary hypoperfusion but rather with a more chronic, structural tissue damage. Recent investigations have revealed an association of higher baseline concentrations of cardiac Troponins with cardiovascular risk in asymptomatic individuals [11–13]. Ischemia-modified albumin (IMA) has been initially proposed as a marker for the diagnosis of myocardial ischemia [14,15]. Metabolic changes associated with ischemia result in a biochemical alteration of the N-terminus of albumin, thereby reducing its affinity to transition metals like cobalt. The albumin cobalt binding (ACB) test has been developed to quantify ischemia modified albumin (IMA) in serum by measuring the concentration of unbound cobalt after adding a defined quantity of cobalt to the serum sample [16,17]. However, IMA is not a tissue-specific marker of ischemia. Several authors have suggested that the generation of IMA from serum albumin is the consequence of contact with reactive oxygen species (ROS) [18,19]. Elevated baseline concentrations have been found in individuals with excessive chronic oxidative stress levels, including patients with end-stage renal disease [20], morbid obesity [19], diabetes mellitus [21] or hypercholesterolemia [18] and affected patients appear to be at an increased risk for cardiovascular complications [22–24]. This study aimed to assess the prognostic value of IMA, BNP and hscTNT for long-term occurrence of major adverse cardiovascular events (MACE) and mortality in ambulatory patients with Fontaine stage II peripheral arterial disease. 2. Methods This investigation was designed as a prospective observational study and patients were recruited in the outpatient clinic of the department of vascular surgery of the Medical University Innsbruck. Because of a lack of preliminary data on the predictive value of IMA on the occurrence of MACE in a comparable patient collective, a power calculation could not be performed. This was a hypothesis-generating investigation. 2.1. Patients Sixty-eight men with stable Fontaine stage II PAD participated in this study. The study protocol has been described previously [25,26]. All patients underwent constant-load treadmill testing at 3 km/h and 12% inclination, to maximum walking distance. Patients with symptomatic CAD, acute myocardial infarction (AMI) within the previous three months and exercise limiting orthopedic or respiratory diseases as well as patients receiving digoxin were not included. All patients included had a normal or only mildly reduced renal function with a glomerular filtration rate of N50 mL/min/1.73 m 2 as calculated by the MDRD-formula (Modification of Diet in Renal Disease; 186 × (Creat / 88.4) − 1.154 × (Age) − 0.203). Body mass index (BMI) was calculated as weight (kg) / height (m)2. During annual visits in the outpatient clinic, participants were monitored for the occurrence of major adverse cardiovascular events (fatal and non-fatal myocardial infarction, coronary artery intervention, coronary artery bypass graft, stroke). Patients who missed their appointment were contacted via telephone and a separate appointment was scheduled. Data of deceased patients were crosschecked with the hospitals' patient documentation system. The study protocol was approved by the institutional research ethics committee and all participants gave written informed consent for participation in the study. Performance of the study complied with the
World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects and/or animals. 2.2. Sample acquisition and handling Before the start of exercise, a short 18-gauge venous catheter (Venflon™, Viggo-Spectramed, Helsingborg, Sweden) was inserted into either the antecubital cephalic vein or the cubital vein of the right arm. Blood was directly drawn into serum and lactate tubes, respectively (S-Monovette®, Sarstedt AG & Co., Nuremberg, Germany) before exercise (=baseline) and 5, 10 and 30 min after cessation of treadmill testing, respectively. The tubes were immediately stored on crushed ice and processed within 60 min by centrifugation at 2500 ×g for 15 min and stored of 500 μL aliquots at − 80 °C. The samples were shipped to the Clinical Institute of Medical and Chemical Laboratory Diagnostics at the Medical University of Graz. N-terminal pro-B natriuretic peptide (NT-proBNP) concentrations were determined with an Elecsys 2010 instrument (Roche Diagnostics, Mannheim, Germany), with a lower detection limit of 5 ng/L and a cutoff value of 125 ng/L. According to the manufacturer the interassay coefficient of variation (CV) was 4.5% for 200 ng/L and 3.6% for 4002 ng/L of quality control material and we found CVs of 3.10% and 2.05% for low and high quality control material, respectively. Serum IMA was measured using the albumin cobalt binding test (Ischemia Technologies Inc., Denver, CO) on a Roche Modular P instrument (Roche Diagnostics, Basel, Switzerland). According to the manufacturer, normal values determined in a population of 283 healthy individuals ranged from 52 to 116 kU/L, resulting in a cut-off value of 85 kU/L (95th percentile). The total interassay CV was 2.8% for quality-control material (mean of three control materials with different concentrations) as determined in the laboratory (Medical University of Graz). Due to the previously described interdependency of IMA and serum albumin, calculation of an IMA/albumin coefficient (kU/g) was performed. Serum albumin was measured with the BCG (bromocresol green) method using enzymatic reagents and standards from Roche. According to the manufacturer the interassay CV for albumin measurements was 1.7%. High sensitive Troponin T (hsTnT) was measured on an Elecsys system (Roche Diagnostics, Mannheim, Germany) using the Troponin T hs test and standards from Roche. The manufacturer reported the interassay CV as 2.6% for 27.9 ng/L and 1.7% for 2049 ng/L of quality control material and we found CVs of 2.96% and 2.12% for low and high quality control material, respectively. According to the manufacturer, the limit of blank is 3 ng/L and 5 ng/L is given as detection limit. The upper reference value was set at 14 ng/L, reflecting the 99th percentile of normal values determined in a population of 546 healthy individuals. 2.3. Statistics The skewed statistical distribution of baseline biomarker concentrations at baseline prompted non-parametric tests (Mann–Whitney-U) for comparison of medians. Quantitative changes of serum concentrations of biomarkers after treadmill testing were analyzed as raw and percentile change scores. Kaplan–Meier-curves (log-rank test) were used for survival analysis of halves (percentiles) of biomarker concentrations. In order to adjust for potential confounders, multivariable proportional hazard models (Cox Regression) including major cardiovascular risk factors (CVRF; age, body mass index (BMI), smoking, diabetes, hypertension, hypercholesterolemia, history of cardiovascular disease) were used to estimate hazard ratios (HR; 95% confidence interval (CI)) for the occurrence of the primary endpoint. The primary combined endpoint comprised major adverse cardiovascular events. Pairwise comparison of significant biomarkers (NT-pro BNP and IMA) was performed in uniand multivariate proportional hazard models using backward elimination removing non-significant variables with MACE as primary endpoint. A two-sided p-value (P) of less than 0.05 was considered to indicate
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statistical significance. All analyses were performed with the use of SPSS 20 (IBM Inc., Somers, NY 10589, USA.).
Two patients were lost to follow-up and excluded from the analysis. Characteristics and distribution of baseline cardiovascular markers of the 66 patients with complete follow-up are presented in Table 1. The observation period lasted from the date of first sample retrieval until the date of last follow-up (median observation period 43.0 months) for all 66 male patients. After the observation period 12 (18.2%) of 66 patients had suffered from a cardiovascular adverse event (primary endpoint; Table 2). Comparison of medians of selected biomarkers showed significantly different serum concentrations of IMA, the ratio of IMA/Albumin and NT-pro BNP in patients with MACE compared to event-free individuals (Table 3). Noteworthy, in multivariable regression models (Cox Regression) correcting for confounders (age, BMI, smoking, diabetes, hypertension, hypercholesterolemia, history of cardiovascular disease) only IMA was significantly (HR = 1.07, CI 1.01– 1.13; p = 0.029) associated with the primary combined endpoint as shown in Table 4. Post-exercise concentrations and quantitative changes of serum concentrations of biomarkers (raw and percentile change scores) after treadmill testing showed no significant association with
Table 1 Characteristics and distribution of baseline cardiovascular markers of patients (n = 68) with peripheral occlusive disease. Dichotomous
Count
Percent (%)
Risk factors Smoking Diabetes Hypertension Hypercholesterolemia Coronary artery disease Cerebrovascular disease
65 9 40 51 20 9
95.6% 13.2% 58.8% 76.1% 29.4% 13.2%
Medication ASA Clopidogrel Heparin Coumadin β-Blocker ACE-inhibitor
55 12 1 6 15 29
80.9% 17.6% 1.5% 8.8% 22.1% 42.6%
Continuous
Median
IQR
Age (years) BMI Maximum walking distance (meter) IMA (kU/L) Albumin (g/L) IMA/Albumin (kU/g) Hs-cTnT (ng/L) NT-pro BNP (ng/L)
66.0 23.0 185.0 103.7 4.4 2.3 6.7 84.3
10.9 5.0 175.0 10.0 0.4 0.5 5.7 92.5
ASA acetylsalicylic acid, BMI body mass index, hs-cTnT high sensitivity cardiac Troponin T, IMA ischemia modified albumin, IQR interquartile range, MCI myocardial infarction, NT-pro BNP N-terminal pro-B natriuretic peptide.
Table 2 Adverse events.
Cardiovascular event (any) Cerebrovascular event AMI Deceased Stroke, fatal AMI, fatal AMI denotes acute myocardial infarction.
Table 3 Comparison of medians of cardiovascular markers by occurrence of primary endpoint (MACE) of 68 patients with peripheral arterial occlusive disease. p-Value by Mann– Whitney-U test. Marker
3. Results
Count
Percent (%)
12 3 9 5 1 4
18.2% 4.5% 13.6% 7.6% 1.5% 6.1%
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IMA (kU/L) IMA/Albumin (kU/g) hs-cTnT (ng/L) NT-pro BNP (ng/L)
Alive
MACE
p-Value
Median
±SE
Median
±SE
102.5 2.3 6.6 84.6
0.9 0.0 0.7 15.4
11.6 2.6 9.4 270.5
2.4 0.1 1.5 295.9
b0.001 0.001 0.356 0.007
hs-cTnT denotes high sensitivity cardiac Troponin T, IMA ischemia modified albumin, NT-pro BNP N-terminal pro-B natriuretic peptide and SE standard error.
occurrence of the primary endpoint (p N 0.05), as presented in Table 5. Kaplan–Meier analysis demonstrated a significantly higher event-free survival rate after 4 years (96.9% versus 66.7%) in patients with lower IMA baseline concentrations (Fig. 1) as well as a significantly decreased cardiac mortality (0% versus 12.1%; Supplemental Fig. 1). After adjusting for confounders, this correlated to an 11.3 time higher risk of suffering a MACE within the observation period (HR = 11.3, CI 1.3–93.1; p = 0.024; Supplemental Fig. 2). As prognostic marker, IMA showed a better discriminatory power (AUC 0.84 CI 0.70–0.97; PI b 0.001) than Nt-proBNP or hs-cTnT (Fig. 2).
Table 4 Uni- and multivariate analyses estimating the association of cardiovascular biomarkers of 66 patients with cardiovascular adverse events (primary endpoint) within the observation time. p-Value by proportional hazard model (Cox Regression). Multivariable model adjusted for cardiovascular risk factors (age, BMI, smoking, diabetes, hypertension, hypercholesterolemia, history of cardiovascular disease). HR
95.0% CI
p-Value
Lower
Upper
Univariate IMA IMA/Albumin hs-cTnT NT-pro BNP
1.08 4.05 1.05 1.00
1.03 1.41 0.94 1.00
1.13 11.62 1.17 1.00
0.003 0.009 0.394 0.024
Multivariate IMA IMA/Albumin hs-cTnT NT-pro BNP
1.07 3.13 1.04 1.00
1.01 0.95 0.92 1.00
1.13 10.35 1.16 1.00
0.029 0.061 0.562 0.061
CI denotes confidential interval, hs-cTnT high-sensitivity cardiac Troponin T, IMA ischemia modified albumin and NT-pro BNP N-terminal pro-B natriuretic peptide.
Table 5 Multivariable analysis of the effect of exercise-dependent serum changes of cardiovascular markers of 66 patients on MACE (primary endpoint) within the observation time. p-Value by proportional hazard model (Cox Regression). Multivariable model adjusted for cardiovascular risk factors (age, BMI, smoker, diabetes, hypertension, hypercholesterolemia, history of cardiovascular disease). HR
95.0% CI (HR)
p-Value
Lower
Upper
RCS multivariate IMA IMA/Albumin hs-cTnT NT-pro BNP
1.05 1.60 1.38 1.00
0.96 0.15 0.87 0.97
1.15 17.12 2.18 1.03
0.306 0.700 0.172 0.857
PCS multivariate IMA IMA/Albumin hs-cTnT NT-pro BNP
1.05 1.01 1.02 1.02
0.96 0.95 0.99 0.96
1.15 1.07 1.04 1.10
0.278 0.776 0.164 0.511
HR denotes hazard ratio, hs-cTnT high sensitivity cardiac Troponin T, IMA ischemia modified albumin, NT-pro BNP N-terminal pro-B natriuretic peptide.
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Fig. 1. Survival estimate (KM; Kaplan Meier curve) for MACE of patients (n = 66) with PAOD by IMA b median compared to IMA ≥ median. p-Value by log-rank test.
4. Discussion Our data provide strong evidence that in patients with Fontaine stage II PAOD, high concentrations of IMA are a strong and independent predictor of cardiovascular complications and overall mortality after an average follow-up interval of 3.6 years. A multivariable logistic regression analysis adjusted for classic risk factors including age, BMI, smoking status, diabetes, hypertension and history of cardiovascular disease identified IMA at baseline as the only independent predictor of reaching the primary end point.
This is in accordance with recent publications. An association of elevated serum concentrations of ischemia-modified albumin with shortterm mortality has been reported after acute myocardial infarction [27] and following cardiopulmonary resuscitation in cardiac arrest patients [28]. An increased risk of major cardiovascular complications as well as a higher 1-year mortality have been found in patients with increased baseline concentrations of IMA after admission for acute coronary syndrome [22,23] and after acute myocardial infarction [29]. Elevated IMA concentrations have been found in diseases that are associated with excessive chronic oxidative stress levels and also with an increased mortality, including end-stage renal disease [20], morbid obesity [19], diabetes mellitus [21] or hypercholesterolemia [18]. Oxidative stress plays a major role in the onset and progression of atherosclerosis [30]. Reactive oxygen species are produced by diseased endothelial cells as well as smooth muscle cells and increased shear stress can also stimulate superoxide production [30]. If the generation of IMA from serum albumin is the consequence of contact with reactive oxygen species, as has been suggested previously [18,19], IMA might in fact be a serum marker for the severity of systemic atherosclerosis. The potential value of IMA for the prediction of adverse cardiovascular events is supported by two important observations. It is independent from concentration changes induced by moderate exercise, as shown through the treadmill testing. A potential association of stress-induced changes of serum IMA concentrations with future adverse events has previously only been investigated once: Sharma et al. reported a significant association of dobutamine stress test induced increase of IMA ≥ 20 kU/L with cardiac as well as all-cause mortality at 1 year in patients with end-stage renal disease while IMA baseline concentrations were not associated with an increased risk [24]. However, the amount of (cardiac) stress during treadmill exercise is certainly not comparable to dobutamine stress testing, especially in a population of patients with a claudication-limited walking distance. Furthermore, the predictive value of IMA does not seem to be affected by concomitant albumin concentrations, as IMA/Alb ratios have not shown a significant association with the occurrence of the primary outcome. Although initial reports claimed that the ACB test would be
Fig. 2. ROC curves estimating discriminatory power (AUC; area under the curve) of selected biomarkers for occurrence of MACE (primary endpoint). Ideal marker AUC N 0.8. hs-cTnT denotes high sensitivity cardiac Troponin T, IMA ischemia modified albumin and NT-pro BNP N-terminal pro-B Natriuretic Peptide. AUC area under the ROC curve, CI confidential interval. C-Index corresponds to an AUC of 0.5 with no discriminatory power.
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insensitive to serum albumin concentrations [31], previous publications have highlighted a negative correlation of serum albumin and IMA [32–34]. Concerning the predictive value of IMA, this relationship appears to be insignificant. Preoperative NT-proBNP concentrations have also been shown to predict myocardial injury or peri- and postoperative death in patients undergoing major non-cardiac vascular surgery [35,36]. The predictive value of NT-proBNP in patients with peripheral arterial disease has also been investigated previously: Mueller et al. reported a significantly increased risk of death of all causes in patients with serum NT-proBNP concentrations above the median of 213 ng/L in a population of 418 patients with peripheral arterial disease including a proportion of 19% of cases with critical limb ischemia [37]. Although we did find a somewhat increased risk of cardiovascular complications in patients with NTproBNP concentrations above the median of 89.7 ng/L in our population, this was not significant in the multivariable analysis. In any case, comparison of ROC curves for IMA and NT-proBNP showed that IMA baseline concentrations were superior in predicting MACE, independent of the selected cutoff-value. Recent publications pointed to a potential value of cardiac Troponins as prognostic markers in addition to their value for the diagnosis of acute cardiac events: Otsuka et al. have reported an association of low-level increases of hs-cTnT in middle-aged men without overt CAD with the Framingham risk prediction score for coronary artery disease and suggested using hs-cTnT for identification of high-risk patients for the primary prevention of CAD [11]. Recently, it has been shown that elevated baseline concentrations of hs-cTnT are a predictor of cardiovascular mortality not only in patients with established coronary artery disease [12] but also in the general population [13]. However, in our collective of Fontaine stage IIb PAOD patients, we did not find an association of either baseline or post-exercise serum concentrations of hscTnT with cardiovascular complications. A limitation of the current investigation is the relatively low number of patients included. However, the low drop out rate of 2.9% during follow-up underlines the high quality of study performance. Moreover, our study only included male patients with unknown, symptom-free coronary or cerebral artery disease and results cannot be applied to female patients. 5. Conclusions In our population of male patients with Fontaine stage II PAOD with unknown, symptom-free coronary or cerebral artery disease, baseline serum concentration of IMA was a strong independent predictor of cardiovascular adverse events. Thus, IMA might serve as a valuable risk marker for future cardiovascular events and cardiovascular mortality. In addition, baseline IMA concentrations are independent from stressinduced changes and not affected by serum albumin concentrations. Our findings are hypothesis-generating and should be further investigated in a larger patient collective, including female individuals. The potential value of IMA as a serum marker for the severity of systemic atherosclerosis must be assessed in a collective that is well defined as far as coronary artery disease, carotid artery disease as well as PAOD are concerned. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2014.08.031. Acknowledgments The technical assistance of the laboratory staff at the Medical University of Graz is gratefully acknowledged. Roche Diagnostics provided assay reagent sets free of charge but did not assume any other role in the conduct of the study. We thank the Society of Atherosclerosis, Thrombosis and Vascular Biology, ATVB20090501 (Verein ATVB), Vienna, Austria, for financially supporting determinations of ischemia-modified albumin.
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