Tag Archives: hYjeF_N2-15q23

and levuglandins are highly reactive γ-ketoaldehydes formed by oxygenation of arachidonic

and levuglandins are highly reactive γ-ketoaldehydes formed by oxygenation of arachidonic acid in settings of oxidative injury and cyclooxygenase activation respectively. 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 radical-catalyzed lipid peroxidation. Prostaglandin H2 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 E2 and D2. Lipid peroxidation yields a series of prostaglandin H2 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 RO4987655 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 carbon tetrachloride treated rats (7) hyperoxia treated mice (8) septic mice (9) and activation of platelets (10). Levels of γKA adducted proteins are expected to be elevated in a wide variety of conditions previously linked to oxidative injury and inflammation (11-23). While the potent cytotoxicity of γKAs and their ability to induce protein aggregation and to disrupt enzymatic function indicate a strong pathologic potential (24-27) meaningful investigation into the extent to which formation of γKA adducts on proteins contributes to hYjeF_N2-15q23 disease will require methods to selectively reduce the levels of γKA adducts to compete effectively with lysyl residues (28). Figure 1 Schematic of scavenging of γ-ketoaldehyde by pyridoxamine. Highly reactive γ-ketoaldehydes can be formed by two pathways during disease processes. Cyclooxygenases convert arachidonic acid to prostaglandin H2 which rearranges non-enzymatically … One important candidate for an effective γKA scavenger is pyridoxamine (PM) a vitamin B6 vitamer. We previously determined that the reaction rate of γKA with PM to form pyrrole adducts was over 2000 times greater than its reaction rate with 253 (M + 1) 235 (M -H2O). The oxime (2.5 g 10 mmol) was dissolved in acetic acid (15 mL) cooled to 10 °C in a large ice-water bath and RO4987655 stirred with RO4987655 zinc dust (2.6 g) at 10-15 °C for RO4987655 1 h and at room temperature for 1 h. Solid was removed by filtration through a bed of Celite and the filtrate was evaporated. The residue was taken in water (10 mL) and pH RO4987655 raised to 8.5 with 1 M NH4OH. Water was removed and the residue was dissolved in methanol (15 mL) and purified by flash chromatography (10-30% methanol in acetic acid) to white solid; 1.6 g (67%); m.p. 118-120 °C; MS 239 (M + 1) 222 (M – NH2) 151 (222 – C5H11) 136 (151 – CH3). To determine the second order rate constant for pyrrole formation with a model γKA 4 1 mM each of 4-oxo-pentanal and PPM PM or RO4987655 SA were incubated together and measurements carried out as described in (29) except that the reaction buffer was 50 mM phosphate buffer in 1:1 acetonitrile-water. Measurement of HNE and isoketal adduction 10 mM PM 10 mM 479.3 →84.1 30 eV (lysyl-IsoK-lactam); m/z 487.3→84.1 30 ([13C6 15N2]lysyl-IsoK-lactam. Additionally the appropriate SRM for adducts of the particular PM analog was performed as shown in Table 1. In summary precursor masses for the 353.3→309.1 30 eV (F2-IsoP) and 357.3→313.1 30 eV ([2H4]-8-epi-PGF2). Measurement of cyclooxygenase products in platelets Human blood was obtained following a protocol approved by the Institutional Review Board of Vanderbilt University. Washed human platelets were isolated as described previously (42 43 The eluted platelets were counted with a..