Oxidative stress and protein aggregation have been implicated in the pathogenesis of neurodegenerative diseases. The formation of neuroprostanes, isoprostane-like compounds formed from oxidation of docosahexaenoic acid, which is uniquely enriched in the brain, is increased in Alzheimer's disease. We recently identified the formation of a new class of highly reactive gamma-keto aldehydes, neuroketals, in vivo as products of the neuroprostane pathway. Neuroketals adduct to lysine residues of proteins with remarkable rapidity and induce cross-linking. Because neuroketals have either a 1,4-pentadiene or 1,4,7-octatriene side chain structure, we hypothesized that they could undergo further oxidation to form neuroketals with an additional hydroxyl group. Oxidation of docosahexaenoic acid in vitro yielded a series of compounds that were confirmed to be oxidized neuroketals by mass spectrometric analyses. Analysis of oxidized neuroketal adducts during oxidation of docosahexaenoic acid in the presence of lysine revealed the formation of oxidized Schiff base and hydroxylactam adducts. Oxidized hydroxylactam neuroketal-lysyl protein adducts, analyzed after digestion of proteins to individual amino acids, were not detected in nonoxidized rat brain synaptosomes but were readily detected following oxidation of synaptosomes. These studies indicate that neuroketals can undergo further oxidation, which in turn suggests that measurement of only unoxidized neuroketal adducts likely underestimates the amount of neuroketal adducts present in the brain in disorders of oxidant stress.