A Rapid Method for Measurement of the Susceptibility to Oxidation of Low-Density Lipoprotein

Abstract

SUMMARY. Oxidation of low-density lipoprotein (LDL) may be important in the pathogenesis of atherosclerosis. We describe a method which measures the oxidation resistance of LDL isolated by a rapid procedure without added antioxidants. LDL was isolated from heparinized plasma by density gradient ultracentrifugation and desalted by gel filtration. The protein concentration was standardized to 50 mg/L and oxidation was promoted by copper (2 flmollL) at 37°C. The total sample preparation time was 2·5 h. Conjugated diene production was monitored at X= 234 nm with computation of the lag time. LDL oxidation was inhibited by EDT A but not heparin. Albumin inhibited LDL oxidation but only in concentrations greater than 50 mg/L. LDL was stable in frozen plasma (-70°C) for 10 weeks, but unstable in the isolated and desalted state. The lag time for LDL from patients treated with the antioxidant probucol was markedly prolonged compared to normal subjects. Additional key phrases: atherosclerosis; antioxidants; probucol; lipid peroxidationThe 'oxidized low-density lipoprotein (LDL) hypothesis' proposes that oxidation of LDL is a critical step in atherosclerosis.t-' Investigation of this hypothesis is hampered by lack of suitable techniques for studying LDL oxidation in a clinical setting. One approach is to isolate LDL from peripheral blood and measure its susceptibility to oxidation in vitro. In 1989, Esterbauer! reported a method for oxidation of isolated LDL by copper ions with assessment of lipid peroxidation by continuous spectrophotometric monitoring of conjugated dienes. However, this method used lengthy LDL isolation procedures (several days) and antioxidants ethylene diamine tetra-acetic acid (EDT A) and butylated hydroxytoluene (BHT) were added to prevent oxidation occurring during the process of LDL preparation. Several modifications of this technique have been reported.v' but these still involve lengthy LDL isolation times (> 18 h) and use added EDTA as an antioxidant during the isolation procedure. We describe and validate a method in which LDL is isolated very rapidly

Glycation impairs high-density lipoprotein function

“…Malondialdehyde concentrations [thiobarbituric acid-reactive substances (TBARS)] in HDL were measured using the thiobarbituric acid assay as described [30]. Conjugated diene concentrations in HDL preparations were determined by measuring their absorbance at 234 nm [31].…”

Factors Affecting S-Homocysteinylation of LDL Apoprotein B

“…Plasma was prepared by centrifugation at 2000g for 10 min at 4°C. LDLs were isolated by ultracentrifugation according to methods described by Himber et al (17 ) and McDowell et al (18 ). Briefly, we added 0.9 mL of plasma to a centrifugation tube containing KBr (0.4451 g), adjusting the density of plasma to 1.300 g/L.…”

Absorption and antioxidant effects of quercetin from onions, in man

“…Isolation of LDLaHDL and VLDL Plasma was obtained from heparinised blood samples by centrifugation at 1200 g for 10 min at 4 C. LDL was isolated from plasma by rapid ultracentrifugation as described by McDowell et al (1995). HDL and VLDL were separated at the same time; the HDL appears as a clear band just above the plasma and the VLDL was removed by tube slicing (Beckman tube slicer) above the LDL band.…”