LIPOPROTEIN FRACTIONATION IN DEUTERIUM-OXIDE GRADIENTS - A PROCEDURE FOR EVALUATION OF ANTIOXIDANT BINDING AND SUSCEPTIBILITY TO OXIDATION

Oxidative modifications of lipoproteins appear to contribute to their atherogenecity. Very low and low density lipoproteins (VLDL and LDL) a re protected against these modifications by antioxidants that can be i ncorporated in vivo or in vitro into the particles. We describe here u ltracentrifugal procedures for isolation of VLDL and LDL that do not r equire subsequent dialysis or buffer equilibration. Lipoproteins were isolated in buffers with physiological ionic composition prepared in D 2O (deuterium oxide). This allowed measurements of the content of anti oxidants and of the susceptibility to oxidation of the isolated LDL wi thout further manipulations. Conventional ultracentrifugal methods use high salt concentrations and require additional steps to eliminate th em. This introduces uncertainties in the evaluation of antioxidant bin ding and on measurements of their effect on VLDL and LDL oxidation. Wi th the method described, the composition of the isolated VLDL and LDL was indistinguishable from that of fractions isolated with KBr gradien ts. Also, the content of alpha-tocopherol was similar. LDL isolated wi th KBr solutions appeared to bind 20-45 % more of the probucol present in serum than LDL isolated in isotonic solutions prepared with D2O Th is was the case with probucol incorporated into plasma or serum in viv o or in vitro. Five out of seven LDL isolated with the D2O procedure f rom different human sera appeared more resistant to Cu2+-catalyzed oxi dation than those obtained with KBr gradients from the same serum. In addition to the gradient procedure, we also describe a preparative ver sion of the method that can be used with multiple samples.