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Isolevuglandins (isoLGs) are a family of γ-ketoaldehydes, aka isoketals or neuroketals, that are generated by free radical-induced oxidation of polyunsaturated fatty acid-containing lipids. Because of their high reactivity toward ε-amino groups of lysyl residues, isoLGs are found as protein adducts in vivo. Plasma levels of isoLG-derived protein modifications are orders of magnitude higher than levels of the corresponding isoprostane. This suggests that while isoprostanes are rapidly cleared from the circulation, isoLG−protein adducts accumulate over the lifetime of the protein, which can be weeks, and this may provide a dosimeter for oxidant stress. We now confirm the postulated formation of the first D series isoLG, iso[7]LGD2, by free radical-induced oxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine in vitro. We also show that iso[7]LGD2−protein adduct levels in blood are the highest known for an isoLG-derived epitope. They average 30-fold higher than isoLGE2−protein and 3-fold higher than iso[4]LGE2−protein levels. Similarly, iso[7]LGD2-derived epitope levels in oxidized low density lipoprotein are 20 times higher than isoLGE2−protein and five times higher than iso[4]LGE2−protein levels. Previous studies showed that plasma levels of protein-bound E series isoLGs, i.e., isoLGE2 and iso[4]LGE2, are elevated in individuals with atherosclerosis as compared with age-matched controls. Plasma iso[7]LGD2−protein immunoreactivity in individuals with atherosclerosis averages 8.5 ± 3.1 nmol/mL, significantly higher (P = 0.01) than the 3.5 ± 0.1 nmol/mL in healthy controls. Plasma levels of iso[7]LGD2−protein adducts are strongly correlated with iso[4]LGE2− (r = 0.933) and isoLGE2−protein adducts (r = 0.877). This supports the hypothesis that isoLGs are generated in vivo by parallel competing radical-induced pathways.

Free radical oxidation of human plasma low-density lipoprotein (LDL) produces 2-pentylpyrrole epitopes that are generated by reaction of 4-hydroxy-2-nonenal (HNE), a product of lipid oxidation, with protein lysyl residues. The HNE-derived 2-pentylpyrrole (“HNE−pyrrole”) epitopes were detected with an enzyme-linked immunosorbent assay (ELISA) using antibodies (ON−KLH) raised against protein-bound 2-pentylpyrrole obtained by the reaction of 2-oxononanal (ON) with keyhole limpet hemocyanin (KLH). HNE−pyrrole epitopes in human plasma are not associated primarily with LDL protein, apolipoprotein (apo) B, since only 15% of the total HNE−pyrrole immunoreactivity is removed by immunoprecipitation of apo B. The levels of ON−KLH immunoreactivity detected in human plasma were found to be significantly elevated in renal failure and atherosclerosis patients when compared to those in healthy volunteers. HNE−pyrrole immunoreactivity was also detected in atherosclerotic plaques. The highest levels were associated with extracellular connective tissue. Levels of ON−KLH immunoreactivity in human plasma far exceed levels of free HNE, presumably because of the rapid clearance of free relative to protein-bound HNE. Therefore, HNE−pyrrole epitopes provide a more indelible marker of oxidative injury than levels of free HNE.

Free-radical oxidation of human plasma low-density lipoprotein (LDL) produces (carboxyalkyl)pyrrole (CAP) epitopes that were detected with enzyme-linked immunosorbent assays using antibodies raised against keyhole limpet hemocyanin (KLH)-bound 2-(ω-carboxyheptyl)pyrrole (CHP) and 2-(ω-carboxypropyl)pyrrole (CPP). These antibodies exhibit high structural selectivity (<0.5% cross-reactivity) in competitive binding inhibition assays with the corresponding human serum albumin (HSA)-bound pyrroles. No cross-reactivity was detected for HSA-bound 2-pentylpyrrole, an epitope that is generated by a reaction of 4-hydroxy-2-nonenal (HNE) with protein lysyl residues. Oxidation of either arachidonic or linoleic acid in the presence of HSA produced an HNE-derived 2-pentylpyrrole epitope. However, only oxidation of linoleic acid formed HSA-bound CHP, while only oxidation of arachidonic acid generated HSA-bound CPP. Since ester hydrolysis with KOH markedly elevated levels of immunoreactive epitopes detected in oxidized LDL, the CAPs are presumably generated by reactions of oxidized cholesteryl esters, triglycerides, and phospholipids with LDL protein, and only some of these oxidized esters are hydrolyzed, e.g., by phospholipase activity associated with LDL. Protein-bound CHP immunoreactivity was detected in human plasma, and levels are significantly elevated in renal failure and atherosclerosis patients compared with healthy volunteers. This provides the first evidence for the biological occurrence of protein-bound CAPs in vivo and further suggests that free-radical oxidation of polyunsaturated lipids produces hydroxyalkenal carboxylate esters whose γ-hydroxy-α,β-unsaturated aldehyde functionality and reactivity resemble that of HNE.

Free-radical oxidation of human low-density lipoprotein (LDL) produces levuglandin (LG)-protein adducts that were detected with an enzyme-linked immunosorbent assay using LGE2-KLH antibodies which recognize LGE2-derived pyrroles. The level of immunoreactivity increases with time of oxidation and reaches a maximum by 8 h. The yield of pyrrole varies nonlinearly with the level of LG adduction to LDL. At low LG:LDL ratios, such as those detected in oxidized LDL, the reaction of primary amino groups with LGE2 produces mostly non-pyrrole adducts that are not immunoreactive. Concommitant phospholipolysis must occur if the generation of immunoreactive epitopes in LDL involves oxidation of arachidonyl phospholipids. Thus, since a protein adduct prepared from synthetic LGE2-2-lysophosphatidylcholine ester showed, at most, only 0.5% cross-reactivity with the LGE2-KLH antibodies, the epitopes detected in oxidized LDL are almost certainly not protein adducts of LG-phospholipid esters. As expected, hydrolysis of the carboxylic ester in the protein adduct of LGE2-2-lysophosphatidylcholine ester by treatment with phospholipase A2 produced a fully immunoreactive LGE2-protein adduct.

The prostaglandin endoperoxide PGH2 rearranges nonenzymatically to generate prostaglandins and secoprostanoic acid levulinaldehyde derivatives such as PGE2 and levuglandin (LG) E2, respectively. Direct detection of LGE2 in biological samples is complicated because it is rapidly sequestered by covalent adduction to endogenous nucleophiles including proteins, which produces LGE2-derived protein-bound pyrroles. Therefore, to detect LGE2−protein adducts in vivo, antibodies were raised against a covalent adduct of LGE2 with keyhole limpet hemocyanin (KLH). This antigen enabled the production of high-titer antibodies that exhibit minimal cross-specificity and are sensitive for detecting LGE2-derived pyrroles. Although pyrrole yields are low at LG/protein ratios found in vivo, an enzyme-linked immunosorbent assay with the LGE2−KLH antibodies detects LGE2-derived protein-bound pyrrole immunoreactivity in human plasma from specific patient populations. Furthermore, prominent immunocytochemical staining of human brain thin sections revealed the presence of LGE2-derived pyrrole immunoreactivity, especially in the meningeal vessels of some patients. This demonstration of LG−protein adducts in human plasma and vasculature provides the first evidence for the biological occurrence of levuglandins in vivo and further suggests that these antibodies might prove useful in diagnostic and mechanistic studies of various disease conditions.

Previous model studies suggested the formation of lysine-based 2-pentylpyrroles as novel late adduction products formed upon exposure of proteins to the lipid peroxidation product 4-hydroxy-2-nonenal (HNE). Two 2-pentylpyrrole immunogens were prepared, one by treating keyhole limpet hemocyanin (KLH) directly with 4-oxononanal and the other by preformation of 6-(2-pentylpyrrol-1-yl)hexanoic acid from 6-aminocaproic acid and 4-oxononanal, followed by carbodiimide coupling to KLH. Pyrrole content and lysine modification in KLH were assayed independently. Following immunization of rabbits, antibody titer increased and plateaued over a 4 month period. The structural specificity of the IgG fractions of the antisera was evaluated through comprehensive competitive ELISA studies. These antibodies were used to verify the time-dependent appearance of the 2-pentylpyrrole epitope in protein exposed to HNE. Potential advantages of antibodies recognizing “advanced lipid peroxidation end products” over those recognizing "early" HNE adduction products are discussed.