Isoketals and levuglandins are highly reactive gamma-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 alpha-ketoaldehydes formed by
carbohydrate or lipid peroxidation, could also effectively protect
proteins from the more reactive gamma-ketoaldehydes.
Pyridoxamine prevented adduction of
ovalbumin and also prevented inhibition of
RNase A and
glutathione reductase activity by the synthetic gamma-ketoaldehyde, 15-E2-isoketal. We identified the major products of the reaction of
pyridoxamine with the 15-E2-isoketal, including a stable
lactam adduct. Two lipophilic analogues of
pyridoxamine, salicylamine and 5'-O-pentylpyridoxamine, also formed
lactam adducts when reacted with 15-E2-isoketal. When we oxidized
arachidonic acid in the presence of
pyridoxamine or its analogues,
pyridoxamine-isoketal adducts were found in significantly greater abundance than the
pyridoxamine-N-acyl adducts formed by alpha-ketoaldehyde scavenging. Therefore,
pyridoxamine and its analogues appear to preferentially scavenge gamma-ketoaldehydes. Both
pyridoxamine and its lipophilic analogues inhibited the formation of lysyl-levuglandin adducts in platelets activated ex vivo with
arachidonic acid. The two lipophilic
pyridoxamine analogues provided significant protection against H2O2-mediated cytotoxicity in HepG2 cells. These results demonstrate the utility of
pyridoxamine and lipophilic
pyridoxamine analogues 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.