Rapid isolation of low density lipoproteins in a concentrated fraction free from water-soluble plasma antioxidants

Abstract

A rapid method is described for isolation and concentration of plasma low density lipoproteins (LDL) using a Beckman L80 ultracentrifuge equipped with a 70.1 Ti fixed angle rotor. The isolation of LDL achieved by a discontinuous gradient density step (180 min) was followed by a simultaneous purification and concentration step (45 min) using ultrafiltration through a collodium bag under nitrogen. This dialysis/concentration step, in contrast to the standard dialysis techniques in batch or by filtration through short gel columns, prevents oxidation and dilution of the sample. Electrophoresis in agarose and sodium dodecylsulfate-polyacrylamide (SDS-PAGE) gels were used to monitor LDL surface charge, purity, and contamination with plasma proteins. The artifactual oxidation of LDL during isolation and subsequent handling, and thus the ability of LDL preparation for oxidation/antioxidation studies, was assessed by the determination of endogenous hydroperoxides and thiobarbituric acid reactive substances. The dialysis/concentration step by ultrafiltration that allows the obtention of a concentrated and purified LDL preparation was validated by the absence of ascorbate and urate, as measured by HPLC. This method led to LDL preparations free of water-soluble plasma antioxidants that were minimally oxidized and suitable for reliable in vitro LDL oxidation and inhibition studies. The applicability of this methodology was tested by studying the alpha-tocopherol content of LDL in a Portuguese population of university students