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esearch Council of Hungary. A single center, prospective clinical study was performed at the Institute of Cardiology of the University of Debrecen to investigate the relationship between serum sACE2 activity PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19645759 and cardiovascular pathologies, among other parameters. There were three study groups. Healthy individuals without any cardiovascular pathology or medication were recruited with normal cardiac morphology and left ventricular ejection fraction above 50%. A second group of 239 hypertensive patients was established. This group was characterized by preserved ejection fraction besides optimal antihypertensive therapy according to the national guidelines. Circulating ACE2 in Human Heart Failure A third group of 100 patients with severe left ventricular systolic dysfunction with indication of cardiac resynchronization therapy were also enrolled into the study. Patients were selected for CRT according to the current ESC guideline related to pharmaceutical and MedChemExpress SB366791 device therapy of systolic heart failure Till the date 65 patients fulfilled the first visit between 6 and 9 months, so statistical analyses and diagrams contain exclusively their data. Significant mitral regurgitation was present in 22 patients, enabling dP/dt measurements. Examinations were performed at the enrollment, at regular visits or just before and between 6 and 9 months after CRT device implantation. Each visit included physical examination with assessment of New York Heart Association functional stage, echocardiographic measurements and blood sample collection for biochemical measurements. Cardiovascular risk assessment comprised age, sex, hypertension, hypercholesterinaemia, diabetes mellitus or ischaemic cardiomyopathy. NYHA classification was performed by independent clinicians who were not aware of echocardiographic data. Medical reports and medication history were obtained from all patients. upon substrate hydrolysis using a fluorescence microplate reader. Initial enzyme activities were determined from the linear rate of fluorescence increase over the 0120 min time course. The increase in fluorescence was plotted as a function of reaction time and fitted with a linear regression. sACE2 activity was calculated by the equation: sACE2 activity = D where S is the rate of observed increase in fluorescence intensity, k is the change in fluorescence intensity upon the complete cleavage of 0.1 nmol of Mca-APK, and D is the dilution of the serum. 1 unit of fluorescence corresponds to the quantity of enzyme which can degrade 0.1 nmol Mca-APK in 1 hour at 37 uC. The specificity of the sACE2 enzyme activity assay was tested by the specific human sACE2 inhibitor DX600, on a single test sample, where DX600 resulted in a complete inhibition of Mca-APK cleavage. Fits were accepted when r. 0.95. Measurement of serum angiotensin converting enzyme activity Assessment of ACE activity was based on the spectrophotometric measurement of FAPGG hydrolysis . The reaction mixture contained 50 mL of serum, 0.5 mM FAPGG acryloyl]-L-phenylalanyl-glycyl-glycine) substrate, 300 mM sodium chloride, and 25 mM HEPES -1-piperazineethanesulfonic acid) at pH 8.2. Measurement of ACE activity is based on the change in the absorption at 340 nm when FAPGG hydrolyzed to furylacryloyl-L-phenylalaline and glycylglycine. The reaction was performed in 96-well plates. Changes in FAPGG absorbance were detected using a microplate reader. Hydrolysis of FAPGG by ACE was recorded in every 5 minutes at 37Cu. Optical densit