(CARBOXYALKYL)PYRROLES IN HUMAN PLASMA AND OXIDIZED LOW-DENSITY LIPOPROTEINS

Free-radical oxidation of human plasma low-density lipoprotein (LDL) p roduces (carboxyalkyl)pyrrole (CAP) epitopes that were detected with e nzyme-linked immunosorbent assays using antibodies raised against keyh ole limpet hemocyanin (KTH)-bound 2-(omega-carboxyheptyl)-pyrrole (CHP ) and 2-(omega-carboxypropyl)pyrrole (CPP). These antibodies exhibit h igh structural selectivity (<0.5% cross-reactivity) in competitive bin ding inhibition assays with the corresponding human serum albumin (HSA )-bound pyrroles. No cross-reactivity was detected for HSA-bound 2-pen tylpyrrole, an epitope that is generated by a reaction of 4-hydroxy-a- nonenal (HNE) with protein lysyl residues. Oxidation of either arachid onic or linoleic acid in the presence of HSA produced an HNE-derived 2 -pentylpyrrole epitope. However, only oxidation of linoleic acid forme d HSA-bound CHP, while only oxidation of arachidonic acid generated HS A-bound CPP. Since ester hydrolysis with KOH markedly elevated levels of immunoreactive epitopes detected in oxidized LDL, the CAPs are pres umably generated by reactions of oxidized cholesteryl esters, triglyce rides, and phospholipids with LDL protein, and only some of these oxid ized esters are hydrolyzed, e.g., by phospholipase activity associated with LDL. Protein-bound CHP immunoreactivity was detected in human pl asma, and levels are significantly elevated in renal failure and ather osclerosis patients compared with healthy volunteers. This provides th e first evidence for the biological occurrence of protein-bound CAPs i n vivo and further suggests that free-radical oxidation of polyunsatur ated lipids produces hydroxyalkenal carboxylate esters whose gamma-hyd roxy-alpha,beta-unsaturated aldehyde functionality and reactivity rese mble that of HNE.