Abx pentra 400 autoanalyzer

We first isolated LDL lipoproteins from plasma samples by density gradient ultracentrifugation [27 (link),31 (link)] and stored them at -80°C until use. From the values of the participants’ lipid profile, we calculated LDL-C levels according to the Friedewald formula (whenever triglycerides were <300 mg/dL) [27 (link),31 (link)]. We quantified ApoB in an ABX-Pentra 400 autoanalyzer (Horiba ABX) in plasma [27 (link),31 (link)]B. We measured LDL resistance against oxidation (LDL lag time) from the kinetics of formation of conjugated dienes (oxidized lipid forms) in isolated LDL samples in a pro-oxidant environment [27 (link),31 (link)]. We assessed the oxidation of LDL lipoproteins as the equivalents of malondialdehyde per mg/dL of cholesterol in isolated LDL samples [27 (link)]. From the lipid profile values, we calculated an approximation to LDL average size (the LDL-C/ApoB ratio) [27 (link)]. We determined the lipid composition of isolated LDL particles in an ABX-Pentra 400 autoanalyzer (Horiba ABX) and, from these data, we calculated the triglyceride/total cholesterol ratio in isolated LDL samples. Finally, we assessed LDL ex vivo cytotoxicity in a THP-1 monocyte-derived macrophage model as previously described [27 (link)].

Blood samples were collected into EDTA and serum tubes and were centrifuged for 15 min at 1000g at 4°C and 21°C, respectively. Aliquots were immediately frozen in liquid nitrogen and subsequently stored at −80°C until analysis. Plasma glucose, FFA, TAG and total cholesterol were analyzed with standard enzymatic methods (ABX Pentra 400 autoanalyzer, Horiba ABX, Montpellier, France). Plasma glycerol was analyzed with an enzymatic assay (Enzytec Glycerol, Roche Biopharm, Basel, Switzerland) automated on a Cobas Fara spectrophotometric autoanalyzer. Plasma insulin, leptin, and adiponectin concentrations were analyzed with commercially available radioimmunoassay (RIA) kits (Human insulin specific RIA, human leptin RIA, human adiponectin RIA, Millipore Corporation, Billerica, MA). Plasma concentrations of IL‐6 (Meso Scale Discovery, Gaithersburg, MD), apelin‐12 (Phoenix pharmaceuticals Inc., Burlingame, CA), RBP4 (R&D systems, Minneapolis, MN) and Vaspin (Adipogen, San Diego, IL) were determined by ELISA. The apelin‐12 ELISA has 100% cross‐reactivity with human apelin‐12, apelin‐13, and apelin‐36. Serum ACE activity was measured with a standard enzymatic assay (Bühlmann, Basel, Switzerland) while serum nesfatin‐1 (Phoenix pharmaceuticals Inc., Burlingame, CA) concentrations were measured by ELISA.