Analysis of Free Radicals, Radical Modifications and Redox Signalling 1233 Analysis of oxidized and chlorinated lipids by mass spectrometry and relevance to signalling Corinne M. Spickett* 1 and Norsyahida Mohd Fauzi† *School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K., and †Strathclyde Institute of Pharmacy and Biomedical Science, University of Strathclyde, Glasgow G4 0NR, U.K. Abstract Oxidized and chlorinated phospholipids are generated under inflammatory conditions and are increasingly understood to play important roles in diseases involving oxidative stress. MS is a sensitive and informative technique for monitoring phospholipid oxidation that can provide structural information and simultaneously detect a wide variety of oxidation products, including chain-shortened and -chlorinated phospholipids. MS n technologies involve fragmentation of the compounds to yield diagnostic fragment ions and thus assist in identification. Advanced methods such as neutral loss and precursor ion scanning can facilitate the analysis of specific oxidation products in complex biological samples. This is essential for determining the contributions of different phospholipid oxidation products in disease. While many pro-inflammatory signalling effects of oxPLs (oxidized phospholipids) have been reported, it has more recently become clear that they can also have anti-inflammatory effects in conditions such as infection and endotoxaemia. In contrast with free radical-generated oxPLs, the signalling effects of chlorinated lipids are much less well understood, but they appear to demonstrate mainly pro-inflammatory effects. Specific analysis of oxidized and chlorinated lipids and the determination of their molecular effects are crucial to understanding their role in disease pathology. Background The interest in lipids as mediators and signalling molecules goes back many years, and the effects of enzymatically oxidized fatty acid derivatives such as PGs (prostaglandins) and leukotrienes in the cardiovascular system are well known. In parallel, studies of hyperlipidaemia and elevated LDL (low-density lipoprotein) in CVD (cardiovascular disease) led to the understanding that oxidative modification of the LDL contributed to the formation of foam cells and cytotoxic effects, and stimulated studies of the underlying mechanisms [1]. In the late 1990s, it was discovered that oxPAPC [oxidized PAPC (palmitoylarachidonoyl- glycerophosphocholine)] had inflammatory biological activ- ity mimicking that of oxLDL (oxidized LDL) [2], and this marked the start of a new focus on the identification of specific oxPLs (oxidized phospholipids) derived from LDL that could contribute to atherogenesis. These and similar products may also be generated from cell membrane phospholipids during situations where increased oxidant levels occur, such Key words: chlorinated phospholipid, inflammation, mass spectrometry, oxidized phospholipid (oxPL), precursor ion scanning, Toll-like receptor (TLR). Abbreviations used: DNPH, 2,4-dinitrophenylhydrazine; ER, endoplasmic reticulum; ESI, electrospray ionization; HETE, hydroxyeicosatetraenoic acid; HNE, 4-hydroxy-trans-2-nonenal; HO-1, haem oxygenase-1; HOCl, hypochlorous acid; ICAM, intercellular adhesion molecule; IL, interleukin; LDL, low-density lipoprotein; LPS, lipopolysaccharide; MALDI, matrix-assisted laser- desorption ionization; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; MRM, multiple reaction monitoring; NF-κB, nuclear factor κB; NOS, nitric oxide synthase; oxPAPC, oxidized PAPC; oxPL, oxidized phospholipid; PAF, platelet-activating factor; PAMP, pathogen-associated molecular pattern; PAPC, palmitoylarachidonoylglycerophosphocholine; PEIPC, 1-palmitoyl-2-(5,6)-epoxyisoprostane E2-sn-glycero-3-phosphocholine; POVPC, palmitoyl- oxovaleroylglycerophosphocholine; PPAR, peroxisome-proliferator-activated receptor; ROS, reactive oxygen species; SOPC, stearoyloleoylglycerophosphocholine; TLR, Toll-like receptor; TNFα, tumour necrosis factor α. 1 To whom correspondence should be addressed (email c.m.spickett@aston.ac.uk). as inflammation and apoptosis, and are thought to contribute to physiological and pathological processes [3]. The phospholipids containing polyunsaturated fatty acyl chains, such as PAPC, are most vulnerable to oxidative attack, although hypohalites (e.g. HOCl) derived from the phagocyte enzyme myeloperoxidase also react with MUFAs (mono-unsaturated fatty acids), the vinyl ether bond of plasmalogens and the head group ethanolamine. There is an enormous variety of possible products, including full-length oxidation products, chain-shortened phospholipids and the corresponding non-esterified fragments of the oxidized fatty acyl chains, of which the aldehydes malondialdehyde and HNE (4-hydroxy-trans-2-nonenal) are well-known examples [4,5]. oxPL products, both esterified and non- esterified, that contain reactive moieties such as aldehydes or alkenals can also generate adducts with proteins by Schiff base or Michael addition reactions. A good understanding of the role of phospholipid oxidation products in disease requires methods to detect specific oxidation products in biological and clinical samples. Not surprisingly, there are many available methods for detecting a variety of oxidized lipid products, as described previously [4]. As a general rule, the simplicity of the method is inversely proportional either to the specificity, sensitivity or quantity of information available. Some of the most convenient and routinely used methods involve spectrophotometric or fluorimetric assays; these often detect several similar oxidation products [e.g. DNPH (2,4-dinitrophenylhydrazine) reacts with most carbonyl-containing compounds] and are susceptible to interference from compounds that are similar in structure but not products of oxidative lipid damage. Antibody- based methods such as the ELISAs for protein carbonyl Biochem. Soc. Trans. (2011) 39, 1233–1239; doi:10.1042/BST0391233 C  The Authors Journal compilation C  2011 Biochemical Society