Synchron cx7

We sampled the venous blood of all subjects between 8:00 am and 9:00 am. Next, we placed 15 mL of blood in a K₂EDTA-containing tube (BD Vacutainer K₃E) to quantify the blood Cr (BCr) and blood V (BV) levels. We also placed the same amount of blood in an anticoagulant-free tube (BD Vacutainer Trace Element Serum, Heidelberg, Germany) to quantify the serum Cr (SCr) and serum V (SV) levels and into an anticoagulant-free tube (BD Vacutainer, SARSTEDT—Nümbrecht, Germany) for the biochemical parameter analysis. After allowing for 60 min of spontaneous blood clotting, we separated the serum from the blood cells via centrifugation at 3000 rpm/h for 10 min, decanted again, and stored in a metal-free polypropylene tube at −20 °C until SCr and SV quantification. We took special care to avoid any contamination with metals during the blood sampling, storage, and analyses. All chemicals used were of analytical grade for spectroscopy (Merck, Darmstadt, Germany). We quantified trace elements using inductively coupled plasma mass spectrometry [33 (link)]. We used a biochemical analyzer Synchron CX7 (Beckman Coulter Company, Indianapolis, IN, USA), with reagents obtained from Beckman Instrumental, Inc. (Galway, Ireland) to determine the biochemical parameters such as fasting serum glucose and lipid profiles.

Creatinin, urea, uric acid, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate dehydrogenase (GLDH), aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (AP), protein and albumin (ALB) were analyzed using a Multichannel Autoanalyzer (Synchron CX7, Beckmann, Hamburg, Germany) as previously described [32 (link)]. Atrial natriuretic peptide (ANP) and cGMP were extracted and analyzed using commercially available radioimmuno assay kits as described [33 (link)]. BNP levels in the plasma samples were below the detection limit of 0.05 ng/ml of the RIA Kit used in the study (BNP-45 (Rat)—RIA Kit, Phoenix Europe GmbH, Karlsruhe, Germany). Plasma concentrations of osteopontin (OPN) were analyzed using the Quantikine^® Mouse&Rat Osteopontin Immunoassay (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Plasma concentrations of cinaciguat were measured using HPLC (2300 HPLC System, Cohesive Technologies, Thermo Fischer Scientific, Waltham, MA, USA) as described elsewhere [34 (link)].