Lipoprotein fractionation in deuterium oxide gradients: a procedure for evaluation of antioxidant binding and susceptibility to oxidation.

Abstract

Oxidative modifications of lipoproteins appear to contribute to their atherogenicity. Very low and low density lipoproteins (VLDL and LDL) are protected against these modifications by antioxidants that can be incorporated in vivo or in vitro into the particles. We describe here ultracentrifugal procedures for isolation of VLDL and LDL that do not require subsequent dialysis or buffer equilibration. Lipoproteins were isolated in buffers with physiological ionic composition prepared in D2O (deuterium oxide). This allowed measurements of the content of antioxidants and of the susceptibility to oxidation of the isolated LDL without further manipulations. Conventional ultracentrifugal methods use high salt concentrations and require additional steps to eliminate them. This introduces uncertainties in the evaluation of antioxidant binding and on measurements of their effect on VLDL and LDL oxidation. With the method described, the composition of the isolated VLDL and LDL was indistinguishable from that of fractions isolated with KBr gradients. Also, the content of alpha-tocopherol was similar. LDL isolated with KBr solutions appeared to bind 20-45% more of the probucol present in serum than LDL isolated in isotonic solutions prepared with D2O. This was the case with probucol incorporated into plasma or serum in vivo or in vitro. Five out of seven LDL isolated with the D2O procedure from different human sera appeared more resistant to Cu(2+)-catalyzed oxidation than those obtained with KBr gradients from the same serum. In addition to the gradient procedure, we also describe a preparative version of the method that can be used with multiple samples.