8 iso pgf2a

Cholesterol and triglycerides were measured enzymatically (Roche Diagnostics, Basel, Switzerland). HDL-C was estimated after the precipitation of apolipoprotein B with phosphotungstate/magnesium [16 (link)]. LDL-C was calculated after lipoprotein separation [16 (link)]. Plasma glucose was measured by the glucose-oxidase method, HbA1c by HPLC, and insulin by microparticle enzyme immunoassay (Abbott Laboratories, Wiesbaden, Germany). Nitrotyrosine plasma concentration was assayed by enzyme-linked immunosorbent assay (ELISA), recently validated by our laboratory [16 (link)]. Plasma 8-iso-PGF2a (Cayman Chemical, Ann Arbor, Michigan, USA.), ICAM-1 (British Bio-technology, Abington, Oxon, UK) and IL-6 (R&D Systems, Minneapolis, MN, USA), were determined with commercially available kits. GLP-1 (active 7-36) was measured by a radioimmunoassay kit (Peninsula Laboratories, Belmont, CA, USA). FRAP was measured according to Benzie et al. [17 (link)] and TRAP according to Ghiselli et al. [18 (link)].
Endothelial function was evaluated measuring the FMD of the brachial artery [19 (link)]. At the end of each test, the subjects rested quietly for 15 min. Then, sublingual nitroglycerin (0.3 mg) was administered, and 3 min later, the last measurements were performed. Response to nitroglycerin was used as a measure of endothelium-independent vasodilation.

Blood samples were collected by cardiac puncture. Serum glucose was determined by using a Blood Glucose Meter (Ascensia ELITE^®, Bayer, Barcelona, Spain). Serum insulin was measured by means of an Ultra Sensitive Rat Insulin ELISA kit using mouse insulin as standard (Crystal Chem Inc., Chicago, IL, USA). Intra-and inter-assay coefficients of variation (CV) were 5.5% and 4.8%, respectively. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by enzymatic tests (Infinity™ AST or ALT Liquid Stable Reagent Thermo Fisher Scientific, Waltham, MA, USA). Serum adiponectin was quantified by ELISA (Biovendor, Modrice, Czech Republic). Intra-and inter-assay CV were 4.2% and 5.9%, respectively. Circulating levels of 8-isoprostane (8-iso PGF2a), a proposed marker of antioxidant deficiency and oxidative stress, were measured by an immunoenzymatic assay (Cayman Chemicals, Ann Arbor, MI, USA). SAA concentrations were assessed using an ELISA kit (BioSource International Inc., Camarillo, CA, USA). Intra-and inter-assay CV were 5.3% and 8.1%, respectively.