Human apo-lipoprotein B from normal plasma contains oxidised peptides

Oxidation of low density lipoprotein (LDL) may be atherogenic, but radical- initiated oxidation of its apoprotein B-100 (apoB) has been little studied. Transition metal ions iron and copper are candidates for mediating radical oxidation of LDL in vivo. Therefore, we studied the copper-ion-induced oxi dation of apoB in human LDL. Using HPLC methods developed in our recent wor k, we studied the destruction of native and the generation of six oxidised amino acids; we also assessed the release of peptides from the LDL particle by FPLC. We observed time-dependent losses of apoB histidine, lysine and g lycine. Long-lived reactive species, the reductant DOPA, and the oxidant hy droperoxides of valine and leucine (measured as hydroxides after reduction) , were generated. Their relative abundance (mol/mol of parent amino acid) w as DOPA > o- and m-tyrosine > dityrosine, valine-hydroxides, leucine hydrox ides. Low molecular weight fragments were also released from the LDL in a t ime-dependent manner, contained hydroperoxides sensitive to GSH peroxidase, and generated radicals on reaction with iron-EDTA. The fragments contained peptides active in the quinone redox cycling procedure, comprising 0.25% o f the supplied LDL amino acids. Characteristic peptides were present in eac h FPLC fraction containing the fragments, as judged by further HPLC fractio nation. Some fragments were present in the unoxidised LDL preparations, and when these were largely removed by FPLC, copper oxidation could still gene rate fragments, suggesting that those present in the starting material migh t indicate prior oxidation. Concordantly, we found that fresh plasma LDL ap oB contained similar to 3% of total plasma protein-bound oxidised amino aci ds, and with the same relative abundance. We conclude that plasma proteins including apoB are subject to physiological oxidation, similar to that infl icted by copper ions; the latter may contribute to intimal LDL oxidation, w hich could be the source of oxidised plasma apoB. (C) 1999 Elsevier Science Ltd. All rights reserved.