Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins)as products of the isoprostane pathway and characterization of their lysylprotein adducts

Isoprostanes are prostaglandin-like compounds produced by non enzymatic per oxidation of arachidonic acid. The cyclooxygenase-derived endoperoxide, pro staglandin H-2, can undergo rearrangement to highly reactive gamma-ketoalde hyde secoprostanoids (levuglandin E-2 and D-2), We explored whether isopros tane endoperoxide intermediates also rearrange to levuglandin-like compound s (isolevuglandins), Formation of a series of isolevuglandins during oxidat ion of arachidonic acid in vitro was established utilizing a number of mass spectrometric analyses. However, these compounds could not be detected in free form in protein-containing biological systems, which we hypothesized w as due to extremely rapid adduction to amines. This was supported by the fi nding that >60% of levuglandin E-2 adducted to albumin within 20 s, whereas -50% of 4-hydroxynonenal still remained unadducted after 1 h. By utilizing electro spray tandem mass spectrometry, we established that these compound s form oxidized pyrrole adducts (lactams and hydroxylactams) with lysine, F ormation of isolevuglandin-lysine adducts on apolipoprotein B was readily d etected during oxidation of low density lipoprotein following enzymatic dig estion of the protein to single amino acids, These studies identify a novel series of extremely reactive products of the isoprostane pathway that rapi dly form covalent adducts with lysine residues on proteins. This provides t he basis to explore the formation of isolevuglandins in vivo to investigate the potential biological ramifications of their formation in settings of o xidant injury.