In vitro modification of LDL
As described (20), LDL was isolated from plasma of healthy donors by ultracentrifugation at a density cutoff range of 1.019–1.063 g/ml, and then treated with fiber in phosphate-buffered saline (pH 7.4) containing 100 The membrane was dialyzed against μm ethylenediaminetetraacetic acid. Sterilize with Mille-GV filters (Millipore) after dialysis. The protein concentration of LDL was measured using bicinchoninic acid (BCA) protein assay reagent (Pierce). Prepare acrolein-modified LDL by incubating 50 µg of LDL with acrolein (0–1 mm) in 0.1 ml of 50 mm sodium phosphate buffer (pH 7.2) at 37 °C.acrolein

Oxidation of LDL (0.5 mg protein/ml) was performed at 37 °C in air in phosphate-buffered saline (pH 7.4). LDL was dialyzed against phosphate-buffered saline (pH 7.4) to remove EDTA before oxidation with 5 μM Cu2+. Free cholesterol and cholesteryl esters were separated by HPLC on an LC-18 column (particle size 5 μm, 4.6 × 250 mm, Supelco, Tokyo), washed with acetonitrile/2-propanol (30/70, v/v) at a certain flow rate 1.0 mL/min and detected by UV absorption at 210 nm as reported in Ref. (21). Cholesteryl ester hydroperoxide (CEOH) and hydroxide (CEOH) were measured using HPLC at 234 nm with UV detector as previously described (22). α-Tocopherol was analyzed by HPLC with an electrochemical detector (Kotaki, Tokyo) set at +800 mV for detection. LC-18 column (particle size 5 μm, 4.6×250 mm) was used, the eluent was methanol/tert-butanol (90/10, v/v) containing 50 mm NaClO4, and the flow rate was 1.0 ml/min.

The data presented in this article are representative examples of several independent experiments. Although different LDL samples exhibited different lag times and oxidation rates, they exhibited similar patterns of oxidative modification.

Autooxidation of Polyunsaturated Fatty Acids
Iron-catalyzed oxidation of arachidonic acid was performed by combining 2 mM arachidonic acid with 10 μM Fe2+, 1 mM ascorbic acid or 10 μM Fe2+ and 1 mM ascorbic acid in 0.1 ml of 0.1 M sodium phosphate buffer (pH 7.4) under atmospheric oxygen. carried out by incubation.