Measurement of Oxidized LDL Levels

Blood samples were collected via brachial puncture into 5-mL heparin vacuum tubes. The samples were centrifuged at 3000 rpm for 10 min at RT, and sera were separated and stored at −80 °C for further analysis (maximum, 6 months). The plasma levels of oxidized low-density lipoprotein (ox-LDL) were measured by using the LP-CHOLOX test carried out on an automated analyzer (Free Carpe Diem, Diacron International, Grosseto, Italy), using a commercial kit (Diacron International, Grosseto, Italy) according to the manufacturer’s instructions. The LP-CHOLOX test evaluates a class of hydroperoxides derived from the lipid peroxidation, which are mainly represented by oxidized cholesterol. Peroxides are able to promote the oxidation of the ferrous iron (Fe²⁺) to ferric iron (Fe³⁺). The LP-CHOLOX test is based on the spectrophotometric measurement (at 505 nm) of the colored complex developed by the binding between the Fe³⁺ and the thiocyanate. The absorbance values are directly proportional to the lipoperoxides concentrations, and the values are related to specific standard solution (400 μEq/L). Results are expressed in μEq/L, and reference values are: Normal, ≤599 μEq/L; slight alteration, from 600 to 799 μEq/L; moderate alteration, from 800 to 999 μEq/L; strong alteration, ≥1000 μEq/L [54 (link),55 (link)].