Pyridoxamine Analogues Scavenge Lipid-Derived γ-Ketoaldehydes and Protect against H₂O₂-Mediated Cytotoxicity

Abstract

Isoketals and levuglandins are highly reactive γ-ketoaldehydes formed by oxygenation of arachidonic acid in settings of oxidative injury and cyclooxygenase activation, respectively. These compounds rapidly adduct to proteins via lysyl residues, which can alter protein structure/function. We examined whether pyridoxamine, which has been shown to scavenge α-ketoaldehydes formed by carbohydrate or lipid peroxidation, could also effectively protect proteins from the more reactive γ-ketoaldehydes. Pyridoxamine prevented adduction of ovalbumin and also prevented inhibition of RNase A and glutathione reductase activity by the synthetic γ-ketoaldehyde, 15-E 2 -isoketal. We identified the major products of the reaction of pyridoxamine with the 15-E 2 -isoketal, including a stable lactam adduct. Two lipophilic analogs of pyridoxamine, salicylamine and 5'O-pentylpyridoxamine, also formed lactam adducts when reacted with 15-E 2 -isoketal. When we oxidized arachidonic acid in the presence of pyridoxamine or its analogs, pyridoxamine-isoketal adducts were found in significantly greater abundance than the pyridoxamine-N-acyl adducts formed by α-ketoaldehyde scavenging. Therefore, pyridoxamine and its analogs appear to preferentially scavenge γ-ketoaldehydes. Both pyridoxamine and its lipophilic analogs inhibited the formation of lysyl-levuglandin adducts in platelets activated ex vivo with arachidonic acid. The two lipophilic pyridoxamine analogs provided significant protection against H 2 O 2 -mediated cytotoxicity in HepG2 cells. These results demonstrate the utility of pyridoxamine and lipophilic pyridoxamine analogs to assess the potential contributions of isoketals and levuglandins in oxidant injury and inflammation and suggest their potential utility as pharmaceutical agents in these conditions.Highly reactive γ-ketoaldehydes are formed via the cyclooxygenase pathway and by radicalcatalyzed lipid peroxidation. Prostaglandin H 2 , the product of the cyclooxygenase enzyme, rearranges in aqueous solution to form a number of eicosanoids, approximately 20% of which are the γ-ketoaldehydes levuglandin E 2 and D 2 . Lipid peroxidation yields a series of † This work was supported by National Institutes of Health Grants GM42056 (MERIT Award to L.J.R.), AG26119, GM15431 (to J . NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 December 8. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript prostaglandin H 2 isomers that also rearrange to corresponding γ-ketoaldehydes, designated as isoketals (IsoK). These γ-ketoaldehydes (γKAs) react extremely rapidly with the lysyl residues of protein to form stable adducts, including a lysyl-lactam adduct and intermolecular crosslinks (1-4). Levels of γKA adducts significantly increase in pathological conditions including atherosclerosis, end-stage renal disease, and Alzheimer's Disease (5,6). Increased γKA adduct formation has also been characterized in experimental models of oxidative injury and inflammation, including carb...

Posttranslational modification by an isolevuglandin diminishes activity of the mitochondrial cytochrome P450 27A1

“…The data obtained support our previous fi ndings that Lys 358 and Lys 476 are both targets of iso [4]LGE 2 adduction in CYP27A1 ( 23 ). Our results also strengthen interpretations from other reports on isoLG-associated loss of enzyme activity ( 29,41,(54)(55)(56) and demonstrate that posttranslational protein modifi cation by isoLGs is not simply a biomarker but is in fact a mechanism by which oxidation of lipids leads to modulation of enzyme activity. It should be noted, then, that in retinal mitochondria, the targets for isoLG adduction may not be limited to CYP27A1.…”

“…Despite similar reactivity, the enzyme activity. IsoLG-mediated loss of enzyme activity has been reported before ( 29,41,(54)(55)(56), however, the evidence provided were indirect. In the pioneering study of Fukuda et al ( 54 ), isoLG-treated HEK-293 cells and cultured atrial HL-1 myocytes showed reduced electrophysiology recordings, but isoLG modifi cation of the cardiac sodium channels responsible for this dysfunction was not shown.…”

“…In contrast to all previous studies ( 29,41,(54)(55)(56), we seem to be the fi rst to conduct the measurement of enzyme activity and simultaneously correlate the isoLG modifi cation to specifi c locations in the primary sequence. The data obtained support our previous fi ndings that Lys 358 and Lys 476 are both targets of iso [4]LGE 2 adduction in CYP27A1 ( 23 ).…”

“…In the pioneering study of Fukuda et al ( 54 ), isoLG-treated HEK-293 cells and cultured atrial HL-1 myocytes showed reduced electrophysiology recordings, but isoLG modifi cation of the cardiac sodium channels responsible for this dysfunction was not shown. In another study, the enzyme activity of purifi ed RNase A and glutathione reductase declined upon treatment with isoLG in vitro, and inclusion of an isoLG scavenger in the reaction mixture was able to prevent this loss of activity ( 41 ). However, mechanisms underlying this loss were not investigated further.…”

Reactive gamma-ketoaldehydes as novel activators of hepatic stellate cells in vitro

“…In vivo, IsoLGs are formed during inflammation, either through increased formation of prostaglandins and re-arrangement of the PGH 2 intermediate formed by cyclooxygenase [23], or during oxidative stress [24,25]. Consistent with this observation, circulating and tissue levels of IsoLGs adducts are increased in multiple disease conditions associated with both, including end-stage renal disease and atherosclerosis [26], hypertension [27], pulmonary fibrosis [22].…”