Quantification of Oxidized LDL Levels

Blood samples were obtained using the brachial puncture method and placed into heparin vacuum tubes with a 5-mL capacity. After being centrifuged at 3000 rpm for 10 min at room temperature, the sera were isolated and stored at a temperature of − 80 °C for subsequent analysis within a maximum period of 6 months. The measurement of plasma levels of oxidized low-density lipoprotein (ox-LDL) was conducted through the employment of the LP-CHOLOX test on an automated analyzer known as Free Carpe Diem, provided by Diacron International, Grosseto, Italy. This test employed a commercial kit from the same manufacturer and was executed following the provided instructions. The LP-CHOLOX test assesses a category of hydroperoxides originating from lipid peroxidation, primarily comprising oxidized cholesterol. These hydroperoxides have the capability to induce the conversion of ferrous iron (Fe²⁺) to ferric iron (Fe³⁺), thereby promoting oxidation. The LP-CHOLOX test is reliant on a spectrophotometric assessment conducted at a wavelength of 505 nm, measuring the development of a colored complex formed by the interaction of Fe3 + and thiocyanate. The resulting absorbance values exhibit a direct correlation to the concentration of lipoperoxides present, and these values are standardized against a specific solution (400 μEq/L). For all the analyzed samples, the calculated intra- and inter-assay CV% values were below t2.8% indicating the high reliability and precision of the used method. The outcomes are expressed in μEq/L units, with reference ranges established as follows: normal (≤ 599 μEq/L), minor alteration (600 to 799 μEq/L), moderate alteration (800 to 999 μEq/L), and significant alteration (≥ 1000 μEq/L) [19 (link)].