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Current Projects of the Lipid Research Group
For descriptions of current research projects, visit the links below.  For general background information, visit Lipid Oxidation and Disease.  For a brief description of previous work, visit Key Findings from the Lipid Research Group.
Lipid Oxidation and Disease
In view of the ready oxidizability of polyunsaturated fatty acids and our aerobic environment, it is understandable that lipid oxidation plays a key role in human health.  It contributes to normal physiological processes such as the biosynthesis of the hormone-like prostaglandins, thromboxanes, and prostacyclins.  It may be recruited in the immune response or programmed cell death (apoptosis), and contributes to changes associated with aging.  It also is involved in pathological processes such as the ischemia- reperfusion injury associated with heart attacks and stroke, and may contribute, inter alia, to retinal degeneration, Alzheimer's, Batten's (juvenile lipofuscinosis), and Parkinson's diseases (1), as well as amyotrophic lateral sclerosis (2).  Involvement of oxidized phospholipids is suspected in the pathogenesis of several chronic inflammatory diseases such as antiphospholipid antibody syndrome (3), rheumatoid arthritis (4, 5), inflammatory bowel disease (6), and multiple sclerosis (7, 8).  However, our understanding of these processes on a molecular level remains primitive.

The importance and challenge of acheiving a molecular level understanding of these processes is exemplified by the involvement of lipid oxidation in atherogenesis.  Three factors contribute to the complexity of the problem: (a) a mixture of fatty esters gives rise to (b) a myriad of electrophilic oxidation products which (c) form covalent adducts with biological nucleophiles.  Both the oxidized lipids and their protein adducts may have biological activities that promote atherogenesis.  Thus, substantial evidence now suggests that atherosclerotic plaques form when monocytes are recruited into the arterial intima to become macrophages where they become foam cells by accumulating large amounts of oxidized low-density lipoprotein (oxLDL) (9, 10).  LDLs are detergent-like globules which transport cholesterol and lipids in blood.  Their outer hydrophilic shell is composed mainly of phospholipids, free cholesterol, and a protein (apoB-100), while their lipophilic core contains cholesterol esters, and triglycerides.  Prominent during the oxidation of LDL is the disappearance of linoleic acid (LA) and arachidonic acid (AA) esters and the formation of numerous reactive oxidation products, many of which attach themselves to the LDL protein.  While the biological importance of oxLDL is known, the molecular details of the LDL to oxLDL transformation are not fully understood.  In fact, "oxLDL is not a single defined chemical entity.  Depending on the extent of oxidation and on the various oxidized products generated, it may show a broad spectrum of biological effects" (11). 

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(1)
Clausen, J. (1984) Demential Syndromes and the lipid metabolism. Acta Neurol. Scand. 70 345-355.
(2)
Davies, K. J. A. (1995) Oxidative stress; the paradox of aerobic life. Biochem. Soc. Symp. 61 1-31.
(3)
Höekkö, S., Miller, E., Dudl, E., Reaven, P., Curtiss, L. K., Zvaifler, N. J., Terkeltaub, R., Branch, D. W., Palinski, W. and Witzum, J. L. (1996) Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids. Recognition of cardiolipin by monoclonal antibodies to epitopes of oxidized low density lipoprotein. J. Clin. Invest. 98 815-825.
(4)
Mapp, P. I., Grootveld, M. C. and Blake, D. R. (1995) Hypoxia, oxidative stress and rheumatoid arthritis. Med. Bull. 51 419-436.
(5)
Winyard, P. G., Tatzber, F., Esterbauer, H., Kus, M. L., Blake, D. R. and Morris, C. J. (1993) Presence of foam cells containing oxidised low density lipoprotein in the synovial membrane from patients with rheumatoid arthritis. Ann. Rheum. Dis. 52 677-680.
(6)
Grisham, M. B. (1994) Oxidants and free radicals in inflammatory bowel disease. Lancet 344 859-861.
(7)
Newcombe, J., Li, H. and Cuzner, M. L. (1994) Low density lipoprotein uptake by macrophages in multiple sclerosis plaques: implications for pathogenesis. Neuropathol. Appl. Neurobiol. 20 152-162.
(8)
Toshniwal, P. K. and Zarling, E. J. (1992) Evidence for increased lipid peroxidation in multiple sclerosis. Neurochem. Res. 17 205-207.
(9)
Brown, M. S. and Goldstein, J. L. (1983) Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Ann. Rev. Biochem. 52 223-261.
(10)
Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C. and Witztum, J. L. (1989) Beyond cholesterol.  Modifications of low density lipoprotein that increase its atherogenicity. N. Eng. J. Med. 320 915-924.
(11)
Parthasarathy, S., D.Steinberg and L.Witztum, J. (1992) The role of oxidized low-density lipoproteins in the pathogenesis of atherosclerosis. Annu. Rev. Med. 43 219-25.
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Key Findings from the Lipid Research Group
Index: Levuglandins / Isolevuglandins / Carboxyalkylpyrroles / Hydroxyoxoalkenoyl Phospholipids
Levuglandins
An unconventional approach led to the Lipid Research Group's discovery of many biologically important products of lipid oxidation.  Rather than activity-directed isolation of molecules from biological sources, they used basic research and chemical logic to predict the structures and properties of the naturally occurring molecules.  Synthesis in the laboratory then facilitated the subsequent detection of the molecules in biological samples.  This modus operandi was especially effective for the Group's discovery of levuglandins (LGs).  In 1977, basic research on the chemistry of the prostaglandin endoperoxide (PGH) intermediates in the biosynthesis of the human hormone-like prostaglandins, prostacyclins and thromboxanes led us to postulate the existence of LGs (1).  By 1984, the production of LGs by a novel nonenzymatic rearrangement of a prostaglandin endoperoxide was demonstrated (2).  However, LGs are extraordinarily reactive.  Detecting them in biological systems is complicated by their rapid binding with and crosslinking of proteins (3,4) and DNA (5).  LG-based DNA-protein crosslinking is especially toxic to cells because it is repair resistant.  Finally, in 1997, two decades after their existence was postulated, immunological techniques provided the first evidence that LG-protein adducts are present in human blood (6).  Mass spectroscopic studies subsequently revealed additional complexities in the biological chemistry of LGs showing that unstable Schiff-base (7) and pyrrole type protein adducts are generated initially but that these undergo further oxidative modification leading to hydroxylactams (8). 
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(1) Salomon, R. G. and Salomon, M. F. (1977) 2,3-Dioxabicyclo[2.2.1]heptane. The strained bicyclic peroxide nucleus of prostaglandin endoperoxides. J. Am. Chem. Soc. 99 3501-3.
(2) Salomon, R. G., Miller, D. B., Zagorski, M. G. and Coughlin, D. J. (1984) Prostaglandin endoperoxides. 14. Solvent-induced fragmentation of prostaglandin endoperoxides. New aldehyde products from PGH2 and a novel intramolecular 1,2-hydride shift during endoperoxide fragmentation in aqueous solution. J. Am. Chem. Soc. 106 6049-60.
(3) Salomon, R. G., Jirousek, M. R., Ghosh, S. and Sharma, R. B. (1987) Prostaglandin endoperoxides. 21. Covalent binding of levuglandin E2 with proteins. Prostaglandins 34 643-56.
(4) Iyer, R. S., Ghosh, S. and Salomon, R. G. (1989) Levuglandin E2 crosslinks proteins. Prostaglandins 37 471-80.
(5) Murthi, K. K., Friedman, L. R., Oleinick, N. L. and Salomon, R. G. (1993) Formation of DNA-protein cross-links in mammalian cells by levuglandin E2. Biochemistry 32 4090-7.
(6) Salomon, R. G., Subbanagounder, G., O'Neil, J., Kaur, K., Smith, M. A., Hoff, H. F., Perry, G. and Monnier, V. M. (1997) Levuglandin E2-Protein Adducts in Human Plasma and Vasculature. Chem. Res. Toxicol. 10 536-545.
(7) Boutaud, O., Brame, C. J., Salomon, R. G., Roberts, L. J., II and Oates, J. A. (1999) Characterization of the Lysyl Adducts Formed from Prostaglandin H2 via the Levuglandin Pathway. Biochemistry 38 9389-9396.
(8) Brame, C. J., Salomon, R. G., Morrow, J. D. and Roberts, L. J., II (1999) Identification of extremely reactive g-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts. J. Biol. Chem. 274 13139-13146.
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Isolevuglandins
Evidence emerged in 1997 suggesting that a large family of LG isomers, later named isolevuglandins (isoLGs), is generated by free radical-induced lipid oxidation (1).   Often nonenzynmatic transformations, such as the endoperoxide to levuglandin rearrangement, are dismissed as biologically irrelevant.  This was the case for nonenzymatic lipid oxidations that result in the generation of prostaglandin-like products.  First reported in 1967, this chemistry was considered an artifact (2) and completely ignored until it was rediscovered more than two decades later and, most importantly, demonstrated to occur in vivo (3,4).  These prostaglandin isomers, now named isoprostanes, are presumed to be formed from isoprostanoid endoperoxide intermediates (isoPGHs) that are produced through free radical-induced oxidation of lipids.  In contrast with the enzymatic (cyclooxygenase) generation of PGHs from free polyunsaturated fatty acids, the free radical-induced generation of isoPGHs operates predominately upon fatty acid phospholipids.  IsoPGHs are also precursors of isoLGs.  The levels of isoLG-protein adducts present in the blood from patients with atherosclerosis are approximately double those found in healthy individuals (5).  Most notably, the correlation of isoLG-protein adduct levels with cardiovascular disease is stronger than the correlation of classical risk factors including low density lipoprotein, total cholesterol, or age.  Furthermore, it is likely that isoLG-protein adducts accumulate over a period of days or weeks.  In contrast , lipid oxidation products that are not protein-bound are cleared from the circulation within minutes.  Thus, isoLG-protein adduct levels represent a dosimeter for long term exposure to toxic products of lipid oxidation (6). 
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(1) Salomon, R. G., Subbanagounder, G., Singh, U., O'Neil, J. and Hoff, H. F. (1997) Oxidation of Low-Density Lipoproteins Produces Levuglandin-Protein Adducts. Chem. Res. Toxicol. 10 750-759.
(2) Nugteren, D. H., Vonkeman, H. and Dorp, D. A. v. (1967) Non-enzymic conversion of all-cis 8,11,14-eicosatrienoic acid into prostaglandin E1. Rec. Trav. Chim. 86 1237-1245.
(3) Morrow, J. D., Harris, T. M. and L. J. Roberts, I. (1990) Noncyclooxygenase oxidative formation of a series of novel prostaglandins: analytical ramifications for measurement of eicosanoids. Anal. Biochem. 184 1-10.
(4) Morrow, J. D., Hill, K. E., Burk, R. F., Nammour, T. M., Badr, K. F. and L. J. Roberts, I. (1990) A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc. Natl. Acad. Sci. USA 87 9383-9387.
(5) Salomon, R. G., Batyreva, E., Kaur, K., Sprecher, D. L., Schreiber, M. J., Crabb, J. W., Penn, M. S., DiCorletoe, A. M., Hazen, S. L. and Podrez, E. A. (2000) Isolevuglandin-protein adducts in humans: products of free radical- induced lipid oxidation through the isoprostane pathway. Biochim Biophys Acta 1485 225-35.
(6) Salomon, R. G., Kaur, K. and Batyreva, E. (2000) Isolevuglandin-protein adducts in oxidized low density lipoprotein and human plasma. A strong connection with cardiovascular disease. Trends Cardiovasc Med 10 53-9.
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Carboxyalkylpyrroles
Some lipid oxidations cleave the fatty acyl moiety producing small biologically active molecules.  A well-known example of this phenomenon is the production of hydroxynonenal (HNE) through oxidative cleavage of phospholipids containing linoleate or arachidonate (1).  Sayre, et al. discovered that the reaction of HNE with proteins generates pentylpyrrole derivatives (2).  Analogous chemistry would produce carboxyalkyl pyrroles (CAPs) through the reaction of hydroxyoxoalkenoyl phospholipids with proteins. To test this hypothesis, the Lipid Research Group developed immunological tools using samples of CAPs prepared by synthesis in the laboratory (3). Since esterolytic treatment markedly increases the levels of CAPs detectable in human blood, a majority of the CAPs apparently are present as phospholipid esters that are not recognized by the antibodies. Besides providing novel markers of lipid-based oxidative protein damage, the detection of CAPs in biological samples implied the in vivo generation of hydroxyoxoalkenoyl phospholipids. These studies led to pivotal insights into the pathogenesis of age-related macular degeneration (4-6).
(1) Esterbauer, H., Schaur, R. J. and Zollner, H. (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biol. Med. 11 81-128.
(2) Sayre, L. M., Arora, P. K., Iyer, R. S. and Salomon, R. G. (1993) Pyrrole Formation from 4-Hydroxynonenal and Primary Amines. Chem. Res. Toxicol. 6 19-22.
(3) Kaur, K., Salomon, R. G., O'Neil, J. and Hoff, H. F. (1997) Carboxyalkyl Pyrroles in Human Plasma and Oxidized Low Density Lipoproteins. Chem. Res. Toxicol. 10 1387-1396.
(4) J. G. Hollyfield, V. L. Bonilha, M. E. Rayborn, X. Yang, K. G. Shadrach, L. Lu, R. L. Ufret, R. G. Salomon and V. L. Perez "Oxidative damage-induced inflammation initiates age-related macular degeneration", Nat Med 2008, 14, 194-8.
(5) S. L. Doyle, M. Campbell, E. Ozaki, R. G. Salomon, A. Mori, P. Kenna, G. J. Farrar, A. S. Kiang, M. Humphries, E. C. Lavelle, L. A. O'Neill, J. G. Hollyfield and P. Humphries "NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components", Nat Med 2012, 18, 791-8.
(6) F. Cruz-Guilloty, A. M. Saeed, J. J. Echegaray, S. Duffort, A. Ballmick, Y. Tan, M. Betancourt, E. Viteri, G. C. Ramkhellawan, E. Ewald, W. Feuer, D. Huang, R. Wen, L. Hong, H. Wang, J. M. Laird, A. Sene, R. S. Apte, R. G. Salomon, J. G. Hollyfield and V. L. Perez "Infiltration of proinflammatory m1 macrophages into the outer retina precedes damage in a mouse model of age-related macular degeneration", Int J Inflam 2013, 2013, 503725, PMCID 3606733.
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Hydroxyoxoalkenoyl Phospholipids
A group at UCLA proposed that 5-oxovaleryl phosphatidyl choline (OV-PC) is a product of lipid oxidation that has important biological activities. This phospholipid is produced by free radical-induced oxidative cleavage of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine. The Lipid Research Group performed total syntheses that confirmed the structures and biological activities of OV-PC and a derivative, G-PC, that both induce endothelial cells to bind monocytes (1). It is likely that this biological activity contributes to the development of atherosclerotic lesions by promoting migration of monocytes into the subendothelial space where they accumulate large quantities of oxidized low density lipoprotein (oxLDL). The occurrence of OV-PC and G-PC in vivo was indicated by their presence in oxLDL and in fatty streak lesions from cholesterol-fed rabbits. These findings sparked intense current research on the biological roles of oxidized phospholipids and prompted the Lipid Research Group's entry into this area. Their hypothesis that hydroxyoxoalkenoyl phospholipids, HODA-PC and HOOA-PC, are produced upon oxidation of linoleyl and arachidonyl phospholipids, respectively, was recently confirmed by mass spectroscopic and liquid chromatographic comparison with samples prepared by unambiguous total syntheses. Collaborative studies with groups at UCLA and The Cleveland Clinic Foundation, revealed that both of these phospholipids show a variety of important biological activities that will be detailed in forthcoming publications.
(1) Watson, A. D.; Leitinger, N.; Navab, M.; Faull, K. F.; Hörkkö; Witztum, J. L.; Palinski, W.; Schwenke, D.; Salomon, R. G.; Sha, W.; Subbanagounder, G.; Fogelman, A. M.; Berliner, J. A. (1997) 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., J. Biol. Chem., 272, 13597-13607
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