November 2012

Levuglandins: finding lipid superglue in vivo

Levuglandin D2 as named by the International Union of Pure and Applied Chemistry 
Levuglandin D2 as named by the International Union of Pure and Applied Chemistry. Photo from Wikipedia. 

Curiosity-driven basic research on the chemistry of PGH2, the endoperoxide intermediate in the biosynthesis of prostaglandins, uncovered a novel nonenzymatic rearrangement that produces levulinic aldehyde derivatives with prostanoid side chains that we named levuglandins, LGE2 and LGD2 (1). Detecting these oxidized lipids in vivo is complicated by their proclivity to stick like superglue to proteins within seconds. They form pyrroles that incorporate the ε-amino group of lysyl residues and generate DNA–protein or protein–protein crosslinks within minutes.

Levuglandin chart 
Figure. Generation of LGs/isoLGs and their adducts with proteins, phosphatidylethanolamine and salicylamine. Click on the above to see this in greater detail in a PDF.

Detecting LG adducts in vivo
Immunoassays with antibodies raised against protein adducts of LGs generated by chemical synthesis provided evidence for their presence in human and mouse blood and tissues and enabled our discovery that free-radical-induced oxidation of arachidonyl phospholipids in low-density lipoprotein generates LGE2 (2) as well as isomers named isolevuglandins (3). Total LG/isoLG-adduct levels in vivo can be measured by exhaustive proteolysis and liquid chromatography–mass spectrometry quantitation of the excised LG-modified lysine (4). MS analysis revealed that the adducts are mainly lactams and hydroxylactams generated by oxidation of the initial pyrrole adducts (4). LC–MS/MS analysis also detected isoLG-phosphatidylethanolamine adducts in human blood and mouse liver (5).

IsoLG-protein adducts are markers of cumulative oxidative injury
A murine Candida sepsis model of inflammation exhibited a 3.5-fold increase in adducts of plasma proteins after pathogen exposure (6). Unlike lipid markers (e.g., isoprostanes), which are rapidly cleared from the circulation, isoLG-protein adducts accumulate. Therefore, like a dosimeter, they provide a cumulative index of oxidative injury. Elevated levels of LGs/isoLGs are found in various disease conditions linked with oxidative stress and inflammation.

Salicylamines selectively trap LGs/isoLGs in vivo
A search for sacrificial primary amines that efficiently trap LGs/isoLGs led to the discovery that ortho-hydroxy benzylamines, salicylamines, are uniquely reactive toward these γ-ketoaldehydes, apparently because the ortho hydroxyl group catalyzes cyclization of an unstable intermediate Schiff base adduct to a pyrrole. By selectively trapping LGs/isoLGs, salicylamines can prevent protein modification in vivo (7). This provides a useful tool for assessing the involvement of LGs/isoLGs in pathology and is a starting point for the development of drugs that neutralize these toxic oxidized lipids.

Characterizing in vivo LG/isoLG modification of specific proteins
The effects of LG/isoLG-protein adduction on protein or cellular function can be studied conveniently in vitro (8). However, understanding the pathological significance of those effects requires knowledge of the specific proteins affected and their levels of modification in disease states. Last year, the detection and characterization of LG/isoLG-protein adducts took a quantum leap forward with the development of LC-MS/MS technology that identified the sites of modification, e.g., the isoLG-modified tryptic peptide AVLKETLR, in a mitochondrial protein, Cyp27A1, extracted from human retina (9).

LGs and isoLGs are among the most potent naturally occurring crosslinking agents. The remaining challenges for understanding the biological significance of LGs and isoLGs include characterizing the structures and biological consequences of LG/isoLG-induced crosslinking. Protein–protein crosslinks may contribute to the disease-related formation and accumulation of protein aggregates. DNA-protein crosslinks could influence gene expression under conditions of oxidative stress.

  1. 1) Salomon, R.G., et al. J. Am. Chem. Soc. 106, 6049 – 6060 (1984).
  2. 2) Salomon, R.G., et al. Chem. Res. Toxicol. 10, 750 – 759 (1997).
  3. 3) Salomon, R.G., et al. Trends Cardiovasc. Med. 10, 53 – 59 (2000).
  4. 4) Brame, C.J., et al. J. Biol. Chem. 274, 13139 – 13146 (1999).
  5. 5) Li, W., et al. Free Radic. Biol. Med. 47, 1539 – 1552 (2009).
  6. 6) Poliakov, E., et al. FASEB J. 17, 2209 – 2220 (2003).
  7. 7) Davies, S.S., et al. Biochemistry 45, 15756 – 15767 (2006).
  8. 8) Davies, S.S., et al. Subcell. Biochem. 49, 49 – 70 (2008).
  9. 9) Charvet, C., et al. J. Biol. Chem. 286, 20413 – 20422 (2011).

Photo of Robert G. SalomonRobert G. Salomon ( is the Charles Frederick Mabery Professor of Research in Chemistry in the department of chemistry at Case Western Reserve University.

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