Total synthesis of oxidized phospholipids. 3. The (11E)-9-hydroxy-13-oxotridec-11-enoate ester of 2-lysophosphatidylcholine.

A total synthesis of (11E)-9-hydroxy-13-oxotridec-11-enoate ester of 2-lysophosphatidylcholine (HOT-PC) was devised to facilitate identification of this oxidized phospholipid. A lactone, 8-(3-oxo-1H,6H-2-oxinyl)octanoic acid (1), believed to be generated through an intermediate (11E)-9-hydroxy-13-oxotridec-11-enoic acid (HOT), is produced upon autoxidation of linoleic acid. A synthesis of lactone 1 methyl ester was accomplished from HOT involving a novel trans-cis isomerization that is driven to completion by cyclization to a hemiacetal. An alternative route to this carbon skeleton was also achieved that provides the lactone 1 itself.

Total synthesis of 17-isoLevuglandin E4 and the structure of C22-PGF4alpha.

The isolevuglandin 17-isoLGE4 (10-acetyl-11-formyl-14-hydroxynonadeca-4(Z),7(Z),12(E),16(Z)-tetr aenoic acid) is a levulinaldehyde derivative that is expected to be generated during the free radical-induced oxidation of docosahexaenoic acid. A total synthesis was executed to facilitate detection and identification of 17-isoLGE4 in biological samples. Conjugate addition of a higher order vinyl cyanocuprate to a gamma-alkoxy enone achieved the final carbon-carbon bond formation to complete a convergent elaboration of the 17-isoLGE4 carbon skeleton. Attempted construction of the requisite vinyl nucleophile synthon using hydrostannylation of an alkyne was foiled by tri-n-butylstannyl radical-promoted isomerization of a cis to a trans double bond. Derivatization of 17-isoLGE4 with methoxylamine under anhydrous or wet conditions generated bismethoximes of 17-isoLGE4 or the isomerized delta11-17-isoLGE4 respectively. Analysis of the mass spectrum of a bismethoxime-pentafluorobenzyl ester-trimethylsilyl ether derivative of 17-isoLGE4 provided presumptive evidence that an incorrect structure was proposed earlier for C22-PGF4alpha, the only F4-isoprostane which is produced enzymatically. We conclude that the 22-carbon analogue of PGF2alpha, produced from docosahexaenoic acid by a cyclooxygenase from trout gill, does not have the same side chains as 17-isoLGE4. Furthermore, we now propose that mass spectral data reported for "C22-PGF4alpha" support a 14-PGF4alpha structure rather than the 17-PGF4alpha structure suggested previously.

HNE-derived 2-pentylpyrroles are generated during oxidation of LDL, are more prevalent in blood plasma from patients with renal disease or atherosclerosis, and are present in atherosclerotic plaques.

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.

Isolevuglandin-protein adducts in humans: products of free radical-induced lipid oxidation through the isoprostane pathway.

A family of extremely reactive electrophiles, isolevuglandins (isoLGs), is generated in vivo by free radical-induced lipid oxidation and rearrangement of endoperoxide intermediates of the isoprostane pathway. Protein adducts of two different oxidized lipids, isoLGE(2) and iso[4]LGE(2), and the corresponding autoantibodies are present in human blood. Western blot analysis of a polyacrylamide gel electrophoresis gel detects several immunoreactive plasma proteins. Only a minor fraction of the isoLG-protein modifications is associated with low density lipoprotein since mean levels were decreased only 20-22% by immunoprecipitation of apolipoprotein B (apoB). Mean levels of both isoLGE(2) and iso[4]LGE(2)-protein adducts in plasma from patients with atherosclerosis (AS) (n=16) or end-stage renal disease (RD) (n=8) are about twice those in healthy individuals (n=25). These elevated levels are not related to variations in age, total cholesterol or apoB. A linear correlation (r=0.79) between plasma isoLGE(2) and iso[4]LGE(2)-protein adduct levels in all 49 individuals is consistent with a common free radical-induced mechanism for the production of both oxidized lipids in vivo. The correlation is even stronger (r=0.86) for patients with AS or RD. That isoLG-protein adduct levels are more strongly correlated with disease than are total cholesterol or apoB suggests an independent defect that results in an abnormally high level of oxidative injury associated with AS and RD.

Isolevuglandin-protein adducts in oxidized low density lipoprotein and human plasma: a strong connection with cardiovascular disease.

Isolevuglandins (isoLGs) are extremely reactive gamma-ketoaldehydes that avidly bind covalently with proteins and cause protein-protein as well as DNA-protein crosslinking. IsoLG-protein adducts are generated upon oxidation of low-density lipoprotein (LDL), and may contribute to atherogenesis since such adducts cause recognition and endocytosis of the modified LDL by macrophage cells. Levels of isoLG-protein adducts in human blood plasma are more closely correlated with disease than are the classical risk factors LDL or total cholesterol. We review the basic research that eventuated in the discovery of isoLGs and describe what is known about their natural occurrence and close connection with cardiovascular disease.

Leukocytes utilize myeloperoxidase-generated nitrating intermediates as physiological catalysts for the generation of biologically active oxidized lipids and sterols in serum.

The initiation of lipid peroxidation and the concomitant formation of biologically active oxidized lipids and sterols is believed to play a central role in the pathogenesis of inflammatory and vascular disorders. Here we explore the role of neutrophil- and myeloperoxidase (MPO)-generated nitrating intermediates as a physiological catalyst for the initiation of lipid peroxidation and the formation of biologically active oxidized lipids and sterols. Activation of human neutrophils in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide (nitrogen monoxide, NO) metabolism, generated an oxidant capable of initiating peroxidation of lipids. Formation of hydroxy- and hydroperoxyoctadecadienoic acids [H(P)ODEs], hydroxy- and hydroperoxyeicosatetraenoic acids [H(P)ETEs], F(2)-isoprostanes, and a variety of oxysterols was confirmed using on-line reverse phase HPLC tandem mass spectrometry (LC/MS/MS). Lipid oxidation by neutrophils required cell activation and NO(2)(-), occurred in the presence of metal chelators and superoxide dismutase, and was inhibited by catalase, heme poisons, and free radical scavengers. LC/MS/MS studies demonstrated formation of additional biologically active lipid and sterol oxidation products known to be enriched in vascular lesions, such as 1-hexadecanoyl-2-oxovalaryl-sn-glycero-3-phosphocholine, which induces upregulation of endothelial cell adhesion and chemoattractant proteins, and 5-cholesten-3beta-ol 7beta-hydroperoxide, a potent cytotoxic oxysterol. In contrast to the oxidant formed during free metal ion-catalyzed reactions, the oxidant formed during MPO-catalyzed oxidation of NO(2)(-) readily promoted lipid peroxidation in the presence of serum constituents. Collectively, these results suggest that phagocytes may employ MPO-generated reactive nitrogen intermediates as a physiological pathway for initiating lipid peroxidation and forming biologically active lipid and sterol oxidation products in vivo.

Characterization of the lysyl adducts formed from prostaglandin H2 via the levuglandin pathway.

Prostaglandin H(2) has been demonstrated to rearrange to gamma-ketoaldehyde prostanoids termed levuglandins E(2) and D(2). As gamma-dicarbonyl molecules, the levuglandins react readily with amines. We sought to characterize the adducts formed by synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins with lysine. Using liquid chromatography/electrospray mass spectrometry, we found that the reaction predominantly produces lysyl-levuglandin Schiff base adducts that readily dehydrate to form lysyl-anhydrolevuglandin Schiff base adducts. These adducts were characterized by examination of their mass spectra, by analysis of the products of their reaction with sodium cyanide, sodium borohydride, and methoxylamine and by the mass spectra derived from collision-induced dissociation in tandem mass spectrometry. The Schiff base adducts also are formed on peptide-bound lysyl residues. In addition, synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins produced pyrrole-derived lactam and hydroxylactam adducts upon reaction with lysine as determined by tandem mass spectrometry. A marked time dependence in the formation of these adducts was observed: Schiff base adducts formed very rapidly and robustly, whereas the lactam and hydroxylactam adducts formed more slowly but accumulated throughout the time of the experiment. These findings provide a basis for investigating protein modification induced by oxygenation of arachidonic acid by the cyclooxygenases.

Protein adducts of iso[4]levuglandin E2, a product of the isoprostane pathway, in oxidized low density lipoprotein.

Levuglandin (LG) E2, a cytotoxic seco prostanoic acid co-generated with prostaglandins by nonenzymatic rearrangements of the cyclooxygenase-derived endoperoxide, prostaglandin H2, avidly binds to proteins. That LGE2-protein adducts can also be generated nonenzymatically is demonstrated by their production during free radical-induced oxidation of low density lipoprotein (LDL). Like oxidized LDL, LGE2-LDL, but not native LDL, undergoes receptor-mediated uptake and impaired processing by macrophage cells. Since radical-induced lipid oxidation produces isomers of prostaglandins, isoprostanes (isoPs), via endoperoxide intermediates, we postulated previously that a similar family of LG isomers, isoLGs, is cogenerated with isoPs. Now iso[4]LGE2-protein epitopes produced by radical-induced oxidation of arachidonic acid in the presence of protein were detected with an enzyme-linked immunosorbent assay. Iso[4]LGE2-protein epitopes are also generated during free radical-induced oxidation of LDL. All of the LGE2 isomers generated upon oxidation of LDL are efficiently sequestered by covalent adduction with LDL-based amino groups. The potent electrophilic reactivity of iso-LGs can be anticipated to have biological consequences beyond their obvious potential as markers for specific arachidonate-derived protein modifications that may be of value for the quantitative assessment of oxidative injury.

New developments in the isoprostane pathway: identification of novel highly reactive gamma-ketoaldehydes (isolevuglandins) and characterization of their protein adducts.

The bicyclic endoperoxide prostaglandin (PG) H2 undergoes nonenzymatic rearrangement not only to PGE2 and PGD2, but also to levuglandins (LG) E2 and D2, which are highly reactive gamma-ketoaldehydes. Isoprostanes (IsoPs) are PG-like compounds that are produced by nonenzymatic peroxidation of arachidonic acid. PGH2-like endoperoxides are intermediates in this pathway. Therefore, we explored whether the IsoP endoperoxides also undergo rearrangement to form IsoLGs. Oxidation of arachidonic acid in vitro resulted in the formation of abundant quantities of compounds that were established to be IsoLGs by using mass spectrometric analyses. However, the formation of IsoLGs could not be detected in biological systems subjected to an oxidant stress. We hypothesized that this was due to extremely rapid adduction of IsoLGs to proteins. This notion was supported by the finding that LGE2 adducted to albumin at a rate that exceeded that of 4-hydroxynonenal by several orders of magnitude: >50% of LGE2 had adducted within 20 s. We therefore undertook to characterize the nature of LG adducts. Using liquid chromatography electrospray tandem mass spectrometry, we established that LGs form oxidized pyrrole adducts (lactams and hydroxylactams) with the epsilon-amino group of lysine. Oxidation of low density lipoprotein resulted in readily detectable IsoLG adducts on apolipoprotein B after enzymatic digestion of the protein to individual amino acids. These studies identify a novel class of ketoaldehydes produced by the IsoP pathway that form covalent protein adducts at a rate that greatly exceeds that of other known aldehyde products of lipid peroxidation. Elucidation of the nature of the adducts formed by IsoLGs provides the basis to explore the formation of IsoLGs in vivo and investigate the potential biological ramifications of their formation in settings of oxidant injury.

Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts.

Isoprostanes are prostaglandin-like compounds produced by non-enzymatic peroxidation of arachidonic acid. The cyclooxygenase-derived endoperoxide, prostaglandin H2, can undergo rearrangement to highly reactive gamma-ketoaldehyde secoprostanoids (levuglandin E2 and D2). We explored whether isoprostane endoperoxide intermediates also rearrange to levuglandin-like compounds (isolevuglandins). Formation of a series of isolevuglandins during oxidation of arachidonic acid in vitro was established utilizing a number of mass spectrometric analyses. However, these compounds could not be detected in free form in protein-containing biological systems, which we hypothesized was due to extremely rapid adduction to amines. This was supported by the finding that >60% of levuglandin E2 adducted to albumin within 20 s, whereas approximately 50% of 4-hydroxynonenal still remained unadducted after 1 h. By utilizing electrospray tandem mass spectrometry, we established that these compounds form oxidized pyrrole adducts (lactams and hydroxylactams) with lysine. Formation of isolevuglandin-lysine adducts on apolipoprotein B was readily detected during oxidation of low density lipoprotein following enzymatic digestion of the protein to single amino acids. These studies identify a novel series of extremely reactive products of the isoprostane pathway that rapidly form covalent adducts with lysine residues on proteins. This provides the basis to explore the formation of isolevuglandins in vivo to investigate the potential biological ramifications of their formation in settings of oxidant injury.

Measurement of oxidation in plasma Lp(a) in CAPD patients using a novel ELISA.

BACKGROUND: LGE2 is produced by the cyclooxygenase- or free radical-mediated modification of arachidonate and is formed during the oxidation of low density lipoprotein (LDL) with subsequent adduction to lysine residues in apo B. We have developed a sensitive enzyme-linked sandwich immunosorbent assay (ELISA) for detection and measurement of LGE2-protein adducts as an estimate of oxidation of plasma LDL and Lp(a). METHODS: The assay employs rabbit polyclonal antibodies directed against LGE2-protein adducts that form pyrroles, and alkaline phosphatase-conjugated polyclonal antibodies specific for apo B or apo (a). It demonstrates a high degree of specificity, sensitivity and validity. RESULTS: Epitopes characteristic for LGE2-pyrroles were quantified in patients with end-stage renal disease (ESRD) that had undergone continuous ambulatory peritoneal dialysis (CAPD) and in a gender- and age-matched control population. In addition to finding that both LDL and Lp(a) levels were elevated in CAPD patients, we also found that plasma Lp(a) but not LDL was more oxidized in CAPD patients when compared to corresponding lipoproteins from healthy subjects. Using density gradient ultra-centrifugation of plasma samples, we found that modified Lp(a) floats at the same density as total Lp(a). CONCLUSIONS: The results of this study demonstrate that oxidation of plasma Lp(a) is a characteristic of ESRD patients undergoing CAPD. This ELISA may be useful for further investigations on oxidation of lipoproteins in the circulation of specific patient populations.

Advanced lipid peroxidation end-products in Alexander's disease.

Rosenthal fibers (RF), intra-astrocytic hyaline inclusions, accumulate in various pathological conditions and are the histological hallmark of Alexander's disease. While the major protein components of RF have been identified, the factors accounting for their pathogenesis, accumulation, and insolubility are largely unknown. In this study, we immunohistochemically examined three cases of Alexander's disease using antibodies to a lysine-derived pyrrole modification arising from 4-hydroxy-2-nonenal, a highly cytotoxic reactive aldehyde produced by lipid peroxidation. In all the cases of Alexander's disease examined, strong immunolabeling of RF by the antibodies to 4-hydroxy-2-nonenal pyrrole adducts were noted. By contrast, age-matched control cases showed no immunoreactivity. These results indicate that modification of protein by lipid peroxidation adducts may play an important role in the formation of RF as well as in the pathogenesis of Alexander's disease. Furthermore, taken together with our previous data indicating advanced Maillard reaction end products in RF, it seems that post-translational modification of RF, initiated by oxidative stress, is critical for both the accumulation and the insolubility of RF, and therefore, by inference, in the pathogenesis of Alexander's disease.

Oxidation of low-density lipoproteins produces levuglandin-protein adducts.

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. Concomitant 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.

Levuglandin E2-protein adducts in human plasma and vasculature.

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.

4-Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer's disease.

Recent studies have demonstrated oxidative damage is one of the salient features of Alzheimer's disease (AD). In these studies, glycoxidation adduction to and direct oxidation of amino acid side chains have been demonstrated in the lesions and neurons of AD. To address whether lipid damage may also play an important pathogenic role, we raised rabbit antisera specific for the lysine-derived pyrrole adducts formed by lipid peroxidation-derived 4-hydroxynonenal (HNE). These antibodies were used in immunocytochemical evaluation of brain tissue from AD and age-matched control patients. HNE-pyrrole immunoreactivity not only was identified in about half of all neurofibrillary tangles, but was also evident in neurons lacking neurofibrillary tangles in the AD cases. In contrast, few senile plaques were labeled, and then only the dystrophic neurites were weakly stained, whereas the amyloid-beta deposits were unlabeled. Age-matched controls showed only background HNE-pyrrole immunoreactivity in hippocampal or cortical neurons. In addition to providing further evidence that oxidative stress-related protein modification is a pervasive factor in AD, the known neurotoxicity of HNE suggests that lipid peroxidation may also play a role in the neuronal death in AD that underlies cognitive deficits.

Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo.

Entry of monocytes into the vessel wall is an important event in atherogenesis. Previous studies from our laboratory suggest that oxidized arachidonic acid-containing phospholipids present in mildly oxidized low density lipoproteins (MM-LDL) can activate endothelial cells to bind monocytes. In this study, biologically active oxidized arachidonic acid-containing phospholipids were produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and analyzed by liquid chromatography and electrospray ionization mass spectrometry in conjuction with biochemical derivatization techniques. We have now determined the molecular structure of two of three molecules present in MM-LDL and Ox-PAPC that induce monocyte-endothelial interactions. These lipids were identified as 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/z 594.3) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (m/z 610.2). These two molecules were produced by unambiguous total synthesis and found to be identical by analytical techniques and bioactivity assays to those present in MM-LDL and Ox-PAPC. Evidence for the importance of all three oxidized phospholipids in vivo was suggested by their presence in fatty streak lesions from cholesterol-fed rabbits and by their immunoreactivity with natural antibodies present in ApoE null mice. Overall, these studies suggest that specific oxidized derivatives of arachidonic acid-containing phospholipids may be important initiators of atherogenesis.

Macrophage recognition of LDL modified by levuglandin E2, an oxidation product of arachidonic acid.

Levuglandin (LG) E2, a secoprostanoic acid levulinaldehyde derivative, is a product of free radical oxidation that forms covalent adducts with lysyl residues on proteins. Treatment of LDL with LGE2 leads to uptake and degradation by mouse peritoneal macrophages. Oxidized LDL, but not acetyl LDL efficiently competed for binding and uptake of LGE2-modified 125I-LDL. This result suggests that LGE2-modified LDL was recognized by a class of scavenger receptor that demonstrated ligand specificity for oxidized LDL but not for acetyl LDL.

(Carboxyalkyl)pyrroles in human plasma and oxidized low-density lipoproteins.

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-(omega-carboxyheptyl)-pyrrole (CHP) and 2-(omega-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 gamma-hydroxy-alpha,beta-unsaturated aldehyde functionality and reactivity resemble that of HNE.

Immunochemical evidence supporting 2-pentylpyrrole formation on proteins exposed to 4-hydroxy-2-nonenal.

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.