Routes to 4-Hydroxynonenal: Fundamental Issues in the Mechanisms of Lipid Peroxidation

Although investigation of the toxicological and physiological actions of α/β-unsaturated 4-hydroxyalkenals has made great progress over the last 2 decades, understanding of the chemical mechanism of formation of 4-hydroxynonenal and related aldehydes has advanced much less. The aim of this review is to discuss mechanistic evidence for these non-enzymatic routes, especially of the underappreciated intermolecular pathways that involve dimerized and oligomerized fatty acid derivatives as key intermediates. These cross-molecular reactions of fatty acid peroxyls have also important implications for understanding of the basic initiation and propagation steps during lipid peroxidation and the nature of the products that arise. Although investigation of the toxicological and physiological actions of α/β-unsaturated 4-hydroxyalkenals has made great progress over the last 2 decades, understanding of the chemical mechanism of formation of 4-hydroxynonenal and related aldehydes has advanced much less. The aim of this review is to discuss mechanistic evidence for these non-enzymatic routes, especially of the underappreciated intermolecular pathways that involve dimerized and oligomerized fatty acid derivatives as key intermediates. These cross-molecular reactions of fatty acid peroxyls have also important implications for understanding of the basic initiation and propagation steps during lipid peroxidation and the nature of the products that arise. HNE 2The abbreviations used are: HNE, 4-hydroxynonenal (4-hydroxy-2E-nonenal); 4-HPNE, 4-hydroperoxynonenal (4-hydroperoxy-2E-nonenal); HPODE, hydroperoxyoctadecadienoic acid; H(P)ETE, hydro(pero)xyeicosatetraenoic acid. was first identified as one of several α,β-unsaturated 4-hydroxyaldehydes formed during CCl4-induced lipid peroxidation in rat liver (1Benedetti A. Comporti M. Esterbauer H. Biochim. Biophys. Acta. 1980; 620: 281-296Crossref PubMed Scopus (658) Google Scholar). The aldehydes detected varied in chain length from 8 to 11 carbons, but the 9-carbon HNE was the most prominent. HNE was recognized as having the greatest toxicological potential among these aldehyde products and over the years has become a paradigm for studying the pathophysiological effects of the cytotoxic products of lipid peroxidation (e.g. Refs. 2Liu Q. Raina A.K. Smith M.A. Sayre L.M. Perry G. Mol. Aspects Med. 2003; 24: 305-313Crossref PubMed Scopus (91) Google Scholar, 3Petersen D.R. Doorn J.A. Free Radic. Biol. Med. 2004; 37: 937-945Crossref PubMed Scopus (334) Google Scholar, 4Leonarduzzi G. Chiarpotto E. Biasi F. Poli G. Mol. Nutr. Food Res. 2005; 49: 1044-1049Crossref PubMed Scopus (125) Google Scholar, 5West J.D. Marnett L.J. Chem. Res. Toxicol. 2006; 19: 173-194Crossref PubMed Scopus (254) Google Scholar). HNE can also trigger signaling events in a physiological context (6Nakashima I. Liu W. Akhand A.A. Takeda K. Kawamoto Y. Kato M. Suzuki H. Mol. Aspects Med. 2003; 24: 231-238Crossref PubMed Scopus (85) Google Scholar, 7Dwivedi S. Sharma A. Patrick B. Sharma R. Awasthi Y.C. Redox Rep. 2007; 12: 4-10Crossref PubMed Scopus (81) Google Scholar), and it is also commonly used as a marker of lipid peroxidation (e.g. Refs. 8Guichardant M. Bacot S. Moliere P. Lagarde M. Prostaglandins Leukot. Essent. Fatty Acids. 2006; 75: 179-182Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar and 9Honzatko A. Brichac J. Picklo M.J. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2007; 857: 115-122Crossref PubMed Scopus (11) Google Scholar). The 4-hydroperoxy analog of HNE (4-HPNE) was recognized originally as an enzymatic product of ω6 fatty acid metabolism in plants (10Gardner H.W. Hamberg M. J. Biol. Chem. 1993; 268: 6971-6977Abstract Full Text PDF PubMed Google Scholar), and more recently in lipid peroxidation, along with the 4-oxo analog, 4-oxononenal (4-oxo-2E-nonenal) (11Lee S.H. Blair I.A. Chem. Res. Toxicol. 2000; 13: 698-702Crossref PubMed Scopus (232) Google Scholar, 12Schneider C. Tallman K.A. Porter N.A. Brash A.R. J. Biol. Chem. 2001; 276: 20831-20838Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 13Sayre L.M. Lin D. Yuan Q. Zhu X. Tang X. Drug Metab. Rev. 2006; 38: 651-675Crossref PubMed Scopus (298) Google Scholar). The 6-carbon analog 4-hydroxyhexenal derived from ω3 fatty acids has been identified as a prominent aldehyde in plant and mammalian tissues (8Guichardant M. Bacot S. Moliere P. Lagarde M. Prostaglandins Leukot. Essent. Fatty Acids. 2006; 75: 179-182Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 14Weichert H. Kolbe A. Wasternack C. Feussner I. Biochem. Soc. Trans. 2000; 28: 850-851Crossref PubMed Scopus (4) Google Scholar). The short chain aldehydes have counterparts in the carboxyl-terminal end of the fatty acid molecule, and for esterified fatty acids, the aldehydes remain bound in phospholipids and other cellular esters or complex lipids. We cannot do justice to the many excellent reviews on the biological role of these products; one review appears as part of this minireview series (15Hazen S.L. J. Biol. Chem. 2008; 283: 15527-15531Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The fundamentals of autoxidation or lipid peroxidation were unraveled in the classical studies of Bolland (16Bolland J.L. Quart. Rev. 1949; 3: 1-21Crossref Scopus (229) Google Scholar), Bateman (17Bateman L. Quart. Rev. 1954; 8: 147-167Crossref Scopus (236) Google Scholar), and colleagues at the British Rubber Producers' Research Association. Their work established the free radical nature of the chain reaction and defined the three well recognized stages of the process as initiation, propagation, and termination (Fig. 1, upper). Notably, in this view of the overall process, the propensity for intermolecular cross-linking achieves a prominence mainly in the later stages as polymerization eventually ensues. The lower half of Fig. 1 serves to introduce two themes that appear later in this review. In these perspectives of lipid peroxidation, intermolecular reactions of product conjugated diene hydroperoxides are implicated at earlier stages of the process, during initiation and propagation. We refer to these alternative paradigms here because they can have a major impact on understanding the main routes to HNE formation. In particular, there is experimental evidence for the formation of dimers in both early phases of autoxidation. These dimeric fatty acids are especially unstable and fragment to produce new radicals, hence their relevance to initiation, and they also appear to readily fragment to aldehydic cleavage products, hence their relevance to the sources of HNE. The cleavage of a fatty acid carbon chain is not a particularly facile chemical reaction, so some special activation is required to promote the ensuing formation of aldehydes. Identifying the basis of this activation has represented a main hurdle in understanding the overall mechanism. An essential part of the activation process involves reaction of the fatty acid with molecular oxygen, but the number of O2 molecules involved and exactly how they react has not been settled. Enzymatic reactions could provide some clues to the mechanism. In particular, the plant fatty acid hydroperoxide lyase reaction offers a plausible hypothesis for a route to 4-HNE (Fig. 2). Plant 9-hydroperoxide lyase (cytochrome P450 CYP74C) cleaves the linoleic acid 9-hydroperoxide (9-HPODE) to two aldehydes, one being 3Z-nonenal. In observing the facile oxidation of 3Z-nonenal to 4-HPNE, we were prompted to propose a non-enzymatic pathway consisting of autoxidative formation of 9-HPODE from linoleic acid, followed by a Hock-type cleavage to give 3Z-nonenal, which undergoes rapid oxidation into 4-HPNE (12Schneider C. Tallman K.A. Porter N.A. Brash A.R. J. Biol. Chem. 2001; 276: 20831-20838Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). It was shown that a route via 3Z-nonenal could account for HNE production in plant tissues (18Noordermeer M.A. Feussner I. Kolbe A. Veldink G.A. Vliegenthart J.F.G. Biochem. Biophys. Res. Commun. 2000; 277: 112-116Crossref PubMed Scopus (30) Google Scholar). We surmised that the acid-catalyzed Hock cleavage could be achieved by the fatty acid carboxylate or other acidic moieties present under conditions of lipid peroxidation. (A short synopsis on the Hock rearrangement is given in supplemental Fig. 1.) Studies with chiral hydroperoxides established the existence of at least two separate routes to 4-H(P)NE (12Schneider C. Tallman K.A. Porter N.A. Brash A.R. J. Biol. Chem. 2001; 276: 20831-20838Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Starting with 9S- and 13S-HPODE as model substrates for autoxidation reactions in a thin lipid film, analysis of the 4-hydroperoxy group showed that 4-HPNE derived from 9S-HPODE was racemic, as expected in the pathway through 3Z-nonenal. In contrast, 4-HPNE derived from 13S-HPODE largely retained the original S-configuration of the starting hydroperoxide. To account for this, we invoked formation of a 10,13-diHPODE intermediate, followed by Hock cleavage between C-9 and C-10 (12Schneider C. Tallman K.A. Porter N.A. Brash A.R. J. Biol. Chem. 2001; 276: 20831-20838Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 19Schneider C. Porter N.A. Brash A.R. Chem. Res. Toxicol. 2004; 17: 937-941Crossref PubMed Scopus (54) Google Scholar). Two groups have synthesized these putative intermediate diHPODEs with a view to testing their intermediacy in HNE production (20Schneider C. Boeglin W.E. Yin H. Stec D.F. Hachey D.L. Porter N.A. Brash A.R. Lipids. 2005; 40: 1155-1162Crossref PubMed Scopus (47) Google Scholar, 21Zhang W. Sun M. Salomon R.G. J. Org. Chem. 2006; 71: 5607-5615Crossref PubMed Scopus (30) Google Scholar). Using these materials, we could show that the 10,13-diHPODE dihydroperoxide does not undergo cleavage to 4-HPNE under autoxidative conditions, thus largely disproving a mechanism involving simple dihydroperoxide intermediates, at least as a major factor under mild conditions (20Schneider C. Boeglin W.E. Yin H. Stec D.F. Hachey D.L. Porter N.A. Brash A.R. Lipids. 2005; 40: 1155-1162Crossref PubMed Scopus (47) Google Scholar). Thus, for HPNE formation from 9-HPODE, there is the possibility that carbon chain cleavage is achieved via a Hock rearrangement, whereas from 13-HPODE, an alternative mechanism must apply. The most plausible alternative mechanism, which can be applied equally to 9- or 13-HPODE, involves cross-molecular reactions of peroxyl radicals, as outlined below in this review. The situation gets even more complicated because in addition to routes via radical-dependent hydroperoxide formation, there is also the possibility of peroxides generated via singlet oxidation of fatty acids. This is physiologically relevant in the retina, where all-trans-retinal can act as a photosensitizer (22Gu X. Zhang W. Salomon R.G. J. Am. Chem. Soc. 2007; 129: 6088-6089Crossref PubMed Scopus (13) Google Scholar). Gu et al. (22Gu X. Zhang W. Salomon R.G. J. Am. Chem. Soc. 2007; 129: 6088-6089Crossref PubMed Scopus (13) Google Scholar) investigated such a pathway and showed that model α-hydroperoxy endoperoxides are cleaved into aldehydes upon treatment with stoichiometric amounts of ferrous iron (Fe2+). The ferrous iron was proposed to reduce the hydroperoxide to an alkoxyl radical, which induces subsequent chain cleavage between the hydroperoxide- and endoperoxide-bearing carbons into two aldehydes. The question as to whether this mechanism of cleavage is also relevant for structurally different hydroperoxy endoperoxides that are formed during radical-dependent lipid peroxidation still needs to be investigated. In general, fatty acid hydroperoxy endoperoxides can readily arise from fatty acids containing at least three double bonds, but not readily from linoleic acid, for example (23Frankel E.N. Chem. Phys. Lipids. 1987; 44: 73-85Crossref PubMed Scopus (146) Google Scholar, 24Yin H. Morrow J.D. Porter N.A. J. Biol. Chem. 2004; 279: 3766-3776Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Also, a novel catalyst, vitamin C, was shown to produce a plethora of HNE-related molecules, including 4,5-epoxy-2E-decenal, 4-oxo-2-nonenal, 4-hydroperoxy-2-nonenal, and 4-hydroxy-2-nonenal, via a proposed pathway involving the hydroperoxide-derived alkoxyl radical from 13S-HPODE (25Lee S.H. Oe T. Blair I.A. Science. 2001; 292: 2083-2086Crossref PubMed Scopus (405) Google Scholar). In addition to these possibilities, other more hypothetical possibilities have been suggested for routes involving single fatty acid molecules, and at least part of these mechanistic schemes may still have a role in HNE production (26Porter N.A. Pryor W.A. Free Radic. Biol. Med. 1990; 8: 541-543Crossref PubMed Scopus (196) Google Scholar). A revision to our thinking on mechanism stemmed from the structural analysis of a prominent group of products with intermediate polarity formed during autoxidation of 15S-HETE and 15S-HPETE (27Schneider, C., Boeglin, W. E., Yin, H., Porter, N. A., and Brash, A. R. (2008) Chem. Res. Toxicol., in pressGoogle Scholar). We identified these as epoxyhydroxy and epoxyhydroperoxy derivatives, respectively, in which the original 15S-OH or 15S-OOH of the starting materials remained in place with unaltered chirality, and a new epoxide group replaced one of the double bonds of the conjugated diene system (Fig. 3). This is different in chemistry from synthesis of the group of epoxyhydroxy products that are often termed hepoxilins (28Brash A.R. Yu Z. Boeglin W.E. Schneider C. FEBS J. 2007; 274: 3494-3502Crossref PubMed Scopus (64) Google Scholar). Mechanistically, the epoxidation of the double bond could be accounted for by cross-molecular reaction of a peroxyl radical with a double bond (27Schneider, C., Boeglin, W. E., Yin, H., Porter, N. A., and Brash, A. R. (2008) Chem. Res. Toxicol., in pressGoogle Scholar). A well documented example for the peroxyl radical-dependent dimerization and polymerization of unsaturated molecules is the copolymerization of styrene and oxygen (Fig. 4) (29Mayo F.R. Miller A.A. J. Am. Chem. Soc. 1956; 78: 1023-1034Crossref Scopus (67) Google Scholar, 30Mayo F.R. J. Am. Chem. Soc. 1958; 80: 2465-2480Crossref Scopus (138) Google Scholar). The polymerization is driven by the addition of an initial styrene peroxyl radical to the styrene double bond forming the peroxide bridge between two styrene monomers and an alkyl radical that reacts with oxygen to re-form the peroxyl radical for the next cycle of addition to a double bond (Fig. 4A). “Unzipping” of the copolymer occurs when an alkyl radical reacts back with the peroxide bridge instead of adding a molecule of oxygen (Fig. 4B). In this so-called homolytic displacement reaction, the peroxide is split into two alkoxyl radicals, one of which forms an epoxide, whereas the other induces cleavage between the carbons bearing the alkoxyl radical and the peroxide group to form the two aldehydes benzaldehyde and formaldehyde in a chain reaction. Related chemistry is characterized with other starting materials (31Sugimoto Y. Taketani S. Kitamura T. Uda D. Matsumoto A. Macromolecules. 2006; 39: 9112-9119Crossref Scopus (25) Google Scholar, 32Matsumoto A. Taketani S. J. Am. Chem. Soc. 2006; 128: 4566-4567Crossref PubMed Scopus (46) Google Scholar). The overall outcome of carbon chain cleavage of peroxide-linked polymers into aldehydes triggered further interest in the dimerization or oligomerization of fatty acid derivatives during autoxidation. Involvement in the Initiation of Lipid Peroxidation—Beginning with studies in the 1970s, Makio Morita and co-workers (33Morita M. Fujimaki M. Agric. Biol. Chem. 1973; 37: 1213-1214Crossref Scopus (6) Google Scholar, 34Morita M. Fujimaki M. J. Agric. Food Chem. 1973; 21: 860-863Crossref PubMed Scopus (19) Google Scholar) sought to identify the components in autoxidizing methyl linoleate that would initiate further peroxidation of lipids. Strikingly, they found that in the absence of added transition metals, fatty acid hydroperoxides showed little activity. Instead, the major active component extracted from partially autoxidized linoleate was identified as a class of peroxide distinct from hydroperoxides; these C-O-O-C-linked products, subsequently identified as fatty acid dimers, were potent inducers of lipid peroxidation. The titles of the articles give the gist of the findings: “Non-hydroperoxide Peroxides as Autocatalysts of Lipid Peroxidation” Monocarbonyls in the Autoxidation of Methyl Linoleate” (34Morita M. Fujimaki M. J. Agric. Food Chem. 1973; 21: 860-863Crossref PubMed Scopus (19) Google Scholar). As indicated in the latter title, in the course of the work, the role of these peroxides in the production of aldehydes was recognized. This aldehyde production was studied with some elegant stable isotope techniques (35Morita M. Tokita M. Chem. Phys. Lipids. 1990; 56: 209-215Crossref Scopus (5) Google Scholar, 36Morita M. Tokita M. Chem. Phys. Lipids. 1993; 66: 13-22Crossref Scopus (4) Google Scholar). Although H(P)NE was not mentioned, other interesting aldehydes, including hexanal, 2-octenal, and 2,4-decadienal, important in food technologies, received mechanistic attention. The work culminated in identification of the active components as peroxide-linked dimers containing two hydroperoxy groups (37Morita M. Tokita M. Lipids. 2006; 41: 91-95Crossref PubMed Scopus (25) Google Scholar). Their overall conclusion is that radical generation and aldehyde production are two facets of the same process, sparked by the inherent instability of the dimeric peroxyhydroperoxides. Involvement in Carbon Chain Cleavage to Give HNE-type Aldehydes—The literature on fatty acid cross-chain reactions includes other key studies, the significance of which has been largely unrecognized in the general scientific literature on lipid peroxidation and H(P)NE production. In a series of articles in the 1980s published in Agricultural and Biological Chemistry and Lipids, Miyashita et al. (38Miyashita K. K. T. Agric. Biol. Chem. Scholar, K. K. T. Agric. Biol. Chem. Scholar, K. K. T. Agric. Biol. Chem. Scholar, K. N. K. T. Lipids. Scopus Google Scholar, K. N. K. T. Agric. Biol. Chem. 49: Scholar) studied the polymerization of methyl linoleate during mild conditions of lipid peroxidation, through the lipid at with to reaction. showed that as as products become peroxide-linked dimers are also present as of their articles the formation and structural analysis of the dimeric products K. K. T. Agric. Biol. Chem. Scholar, K. K. T. Agric. Biol. Chem. Scholar, K. N. K. T. Lipids. Scopus Google Scholar). Their studies with a the products of of the dimeric reaction conditions at as identified by K. N. K. T. Agric. Biol. Chem. 49: Scholar). products that retained the carbons of the original linoleate and products and example of the formation of dimeric peroxides and their in aldehyde production is given in Fig. in this starting from are to the styrene in that double bonds with O2 produce that to two aldehydes and an epoxide to the we characterized in autoxidation (27Schneider, C., Boeglin, W. E., Yin, H., Porter, N. A., and Brash, A. R. (2008) Chem. Res. Toxicol., in pressGoogle Scholar). of the and co-workers W.E. E.N. K. J. Am. Chem. Soc. Scopus Google Scholar, E.N. W.E. E. Lipids. PubMed Scopus Google Scholar) the investigation of dimerization to the more unsaturated to the groups (37Morita M. Tokita M. Lipids. 2006; 41: 91-95Crossref PubMed Scopus (25) Google Scholar, K. K. T. Agric. Biol. Chem. Scholar, K. K. T. Agric. Biol. Chem. Scholar, K. N. K. T. Lipids. Scopus Google Scholar, K. N. K. T. Agric. Biol. Chem. 49: Scholar), they in the dimeric by in W.E. E.N. K. J. Am. Chem. Soc. Scopus Google Scholar, E.N. W.E. E. Lipids. PubMed Scopus Google Scholar). they was the instability of the dimers, such that further of the we a key role for intermolecular cross-linking of peroxyl in fatty acid chain in the early stages of lipid peroxidation, this appears to be key to the production of free products such as H(P)NE as well as molecules with or is one in the early of autoxidation. product formation is conjugated dimerization can become This because peroxyl radical addition to a conjugated diene is over reaction with a double a of conjugated is peroxyl cross-linking can with from the unsaturated fatty acid in that in the of conjugated linoleic acid, the chemistry dimerization by reaction of a peroxyl with the In to the of peroxyl addition to conjugated diene Bolland (16Bolland J.L. Quart. Rev. 1949; 3: 1-21Crossref Scopus (229) Google Scholar) that absence of groups in styrene to the of the of In other with there are to with radical addition and this dimerization for generation of aldehydic products to the cross-linking of molecules in and formation of This to be In to potential in we evidence of of molecular shown by and co-workers Lipids. 12: PubMed Scopus Google Scholar, A. Lipids. PubMed Scopus Google Scholar) in the showed that in to autoxidation of in which hydroperoxides are the main products, when the fatty acids were as a on to a biological the main products were simple linoleic and Lipids. 12: PubMed Scopus Google Scholar). The same was not with It was that this of epoxide product in the of to of the A. Lipids. PubMed Scopus Google Scholar). The relevance of this reaction to oxidation as well as the potential dimerization to be investigated. Notably, dimerization is well characterized with methyl but there are of for this chemistry with more unsaturated fatty acids and their biological with