Original Contribution PATHWAYS OF PHOSPHOLIPID OXIDATION BY HOCl IN HUMAN LDL DETECTED BY LC-MS ANDREAS JERLICH,* ,1 ANDREW R. PITT, † R. J¨ ORG SCHAUR, ‡ and CORINNE M. SPICKETT* *Department of Immunology, University of Strathclyde, The Todd Centre, Glasgow, United Kingdom; † Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United Kingdom; and ‡ Institute of Biochemistry, University of Graz, Graz, Austria (Received 9 August 1999; Revised 9 December 1999; Accepted 9 December 1999) Abstract—A wealth of evidence now indicates that low-density lipoprotein (LDL) must be modified to promote atherosclerosis, and that this may involve oxidants released by phagocytes. Many studies of oxidative damage in atherosclerosis previously have concentrated on damage by nonhalogenated oxidants, but HOCl is a highly toxic oxidant produced by myeloperoxidase in phagocytes, which is also likely to be important in the disease pathogenesis. Currently some controversy exists over the products resulting from reaction of HOCl with LDL lipids, in particular regarding whether predominantly chlorohydrins or lipid peroxides are formed. In this study LC-MS of phosphatidylcholines in human LDL treated either with HOCl or the myeloperoxidase system was used as a specific method to detect chlorohydrin and peroxide formation simultaneously, and with comparable sensitivity. Chlorohydrin products from lipids containing oleic, linoleic and arachidonic acids were detected, but no hydroperoxides of linoleoyl or arachidonoyl lipids could be observed. This study provides the first direct evidence that lipid chlorohydrins rather than peroxides are the major products of HOCl- or myeloperoxidase-treated LDL phospholipids. This in turn provides important infor- mation required for the study of oxidative damage in vivo which will allow the type and source of oxidants involved in the pathology of atherosclerosis to be investigated. © 2000 Elsevier Science Inc. Keywords—Atherosclerosis, Chlorohydrins, Electrospray ionization mass spectrometry, HPLC, Hypochlorous acid, Lipid peroxidation, Low density lipoprotein, Myeloperoxidase, Free radicals INTRODUCTION It is now well accepted that high plasma low-density lipoprotein (LDL) levels are a key risk factor in cardio- vascular disease, and that oxidized LDL contributes to the pathology of atherosclerosis [1,2]. There has been much interest in determining the precise nature of this oxidation, and identifying the agents responsible for it. Recently attention has focused on the role of phagocytic cells as a major source of reactive oxygen species, in- cluding the neutrophil, which produces large amounts of hypochlorous acid (HOCl) in a reaction catalysed by the highly abundant, heme enzyme myeloperoxidase (MPO). Evidence for the involvement of this enzyme in athero- sclerosis comes from the observation that it is present in an active form in atherosclerotic lesions [3], that HOCl- modified epitopes of LDL have been detected in such lesions using a specific monoclonal antibody [4], and that 3-chlorotyrosine is elevated in LDL from atherosclerotic intima [5]. In fact, chlorinated tyrosine derivatives, which are thought to be specific products of MPO, have been detected during all stages of development of ath- erosclerosis [6]. However, many details regarding the mechanism and site of damage, and molecular events in the pathology remain to be answered. LDL contains both a protein and a lipid component, either of which may be involved in the oxidative damage during atherosclerosis, and consequently a number of studies have investigated the effects of HOCl on these macromolecules. HOCl in the presence of chloride exists in equilibrium with its anion and molecular chlorine [7], and it is known to undergo a variety of oxidation and chlorination reactions with biological molecules. Address correspondence to: Dr. Corinne M. Spickett, Department of Immunology, University of Strathclyde, The Todd Wing, SIBS, 27 Taylor Street, Glasgow G4 0NR, UK; Tel: +44 (141) 548-3827; Fax: +44 (141) 552-6674; E-Mail: c.m.spickett@strath.ac.uk. 1 A.J. is on leave from the Institute of Biochemistry, University of Graz, Graz, Austria. Free Radical Biology & Medicine, Vol. 28, No. 5, pp. 673– 682, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter PII S0891-5849(99)00273-7 673