Heavy Metals (Cadmium, Lead, Mercury, Zinc), Angiogenesis Factors (Endostatin, Angiostatin, VEGF) and Osteopontin in Patients With Coronary Artery Disease

Patients with angiographically verified CAD of different severity were recruited at the Department of Cardiology, Medical University of Vienna. Detailed anamnestic and clinical data was collected incl. cardiovascular risk factor assessment, medication, ECG (electrocardiogram), routine laboratory parameters, echocardiography and all patients underwent an coronary angiography for diagnostic and/or therapeutic reasons on grounds of their underlying disease. The coronary artery system was divided into 17 segments and stenosis grade for each segment was measured. The segments were: left main, proximal/medial/distal LAD (lad left anterior descending artery), ramus circumflex, first and second marginal branch, posterolateral branch, first and second diagonal branch, proximal/medial/distal LCX (lcx left circumflex artery), proximal/medial/distal RCA (right coronary artery), ramus interventricular posterior and stenosis grade for each segment was measured. A simple 3-point-grading system ("Coronary Score") was developed considering both frequency and severity of CAD: 0 points for non-stenosed or only calcified segments, 1 point for each stenosis from <30-<50 %, 2 points for each stenosis from 50-<70 % and 3 points for each stenosis >70 %. Blood samples for determination of ES, AS and VEGF levels were taken at least two days before or after an acute event (angina pectoris, STEMI-ST-elevation myocardial infarction, NSTEM-Non ST-elevation myocardial infarction) or an invasive intervention (angiography). ES, AS and VEGF were analysed in serum, OPN in plasma by ELISA-Enzyme Linked Immunosorbent Assay according to the instructions of the manufacturer.

Echocardiography was performed to evaluate left and right ventricular function (multiple cross-sectional views), valve insufficiency/stenosis/calcification and wall movement disorders.

Hg and Pb levels were measured in full-blood, Cd in urine. The outcrop of full-blood samples for the determination of Pb and Hg was done by 2 ml ultrapure water and 2 ml nitric acid (68% sub-boiled). The sample aliquot was 0,5 ml, backfilling volume 20 ml. The determination of Pb and Cd was performed by ICPMS (inductively coupled plasma mass spectrometry) according to the ÖNORM EN ISO 17294-2. The determination of Hg was done by AFS (atomic fluorescence spectroscopy) according to the ÖNORM EN 17852. The outcrop of urine samples for the determination of Cd was done by 2 ml ultrapure water and 2 ml nitric acid (68% sub-boiled). The sample aliquot was 5 ml, backfilling volume 20 ml. The detection/quantification limits were 0,40/2 μg/l (Pb), 0,067/0,13 μg/l (Hg) and 0,12/0,40 μg/l (Cd). In case the quantification limit was undercut, the following expected amounts were used: Cd: 0,3 μg/l, Pb: 1 μg/l, Hg: 0,1 μg/l. In case of Cd and Hg Human-Biomonitoring (HBM)-I and II levels and in case of Pb reference levels from the German Environmental Agency.

Physical activity of the patients was defined/quantified as non physical activity, walking less 3h/week, walking more than 3 hours/week, sports less than 3 hours/week and sports more than 3 hours/week.

Statistical analysis was done with SPSS 20.0. Continuous and normally distributed data is described by means ± standard deviation (SD) and group differences are tested by independent sample t-test and correlation were calculated using Pearson's correlation coefficient. Continuous data with skew distribution or outliers is described ny median, first and third quartile and minimum and maximum. Group differences are tested by Mann-Whitney-U-test and correlations were calculated using Spearman's correlation coefficient. For data with values below the quantification limit a value below quantification limit was imputed (the same value for all these observations) and the non-parametric Mann-Whitney-U-test was used. All tests are performed two-sided and p-values ≤ 0,05 were considered significant. The protocol was approved by the Ethical Commission of the Medical University of Vienna and informed consent was obtained from patients.