Comparison of Three Methods for Measuring LDL Resistance against Copper-induced Oxidation

[摘要] Oxidation of LDL in the subendothelial space of the arterial wall initiates a series of events leading to enhanced uptake of LDL by scavenger receptors on macrophages and subsequent foam cell formation. Therefore, LDL oxidation is supposed to play a role in the development of atherosclerosis (1)(2).The most uniformly accepted procedure for measuring the resistance of LDL to in vitro oxidation is determination of the lag time (LT) for conjugated diene (CD) formation, initiated by catalytic amounts of transition metal ions (3). Another method is based on the oxidation of parinaric acid (PA), a fluorescent and oxidation-sensitive polyunsaturated fatty acid (FA) probe, incorporated into LDL (4). A third method monitors fluorescence development (FD) during LDL oxidation (5), caused by reaction of protein amino groups with aldehydes generated during decomposition of peroxidized FAs (6).The three methods described above measure different stages of the oxidation process. Oxidation of the PA probe represents one of the earliest stages of oxidation. The formation of CDs represents an intermediate stage of the oxidation process, and finally FD represents a late stage of the oxidation process.The methods for measurement of LDL oxidizability were evaluated with respect to reproducibility and potential for automation. Their relationship with LDL constituents, as determinants of LDL oxidation, was also studied.Subjects were 98 normolipidemic type 2 diabetic patients with good glycemic control (mean hemoglobin A1c, 6.2% ± 1.0%). LDL was isolated by ultracentrifugation between densities 1.019 and 1.063 kg/L, as described elsewhere (7). PA was purchased from Molecular Probes. Other reagents used were obtained from Merck or Sigma.Total cholesterol, free cholesterol, triglycerides, and phospholipids were analyzed using enzymatic test kits (Boehringer Mannheim). The cholesterol ester content of LDL was calculated as total minus free cholesterol. LDL lipid constituents were standardized for LDL protein …

[学科分类] 过敏症与临床免疫学