The Free Radical Oxidation of Polyunsaturated Lecithins NED A. PORTER, RICHARD A. WOLF and HUGO WEENEN, Paul M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27706 ABSTRACT Two unsymmetric polyunsaturated lecithins were allowed to air oxidize and the primary products of autoxidation were isolated and characterized. 1-Palmitic-2-1inoleic-phosphatidylcholine undergoes significant oxidation after 16 hr at room temperature under air, A new phospholipid product may be isolated by reverse phase high pressure liquid chromatography (HPLC) and this HPLC fraction is shown to be made up of lipid hydroperoxides formed by free radical oxidation of the homoconjugated diene of the linoleate component of the lecithin, l-Stearic-2-arachidonic-phosphatidylcholine under- goes a similar oxidation with the arachidonate polyunsaturated functionality being oxidized. The structure of the oxidation products was established by reduction of hydroperoxide with triphenyl- phosphine, snake venom hydrolysis of the C-2 ester, and HPLC analysis of the resulting hydroxy fatty acids or their methyl esters. INTRODUCTION The oxidation of membrane phospholipid has been suggested to be the primary chemical reaction in a variety of pathological events. For example, membrane damage induced by radia- tion (1) and carbon tetrachloride or ethanol poisoning (2-4) has been proposed to be the result of phospholipid destruction by molecular oxygen. In fact, a theory of aging has been proposed that rests, in part, on the free radical oxidation of membrane lipid (5). In light of the potential importance of phos- pholipid oxidation products, it seems remark- able that little effort has been made to purify and chemically characterize these species. Oxi- dation of polyunsaturated phospholipid leads to conjugated diene as detected in the ultra- violet and also to peroxide products (6) as indicated by titration. However, no primary phospholipid oxidation products have as yet been purified and the standard methods of phospholipid purification (silica gel column and thin-layer chromatography) would appear to offer little hope for isolation of these primary oxidation products. Recently, high pressure liquid chromatog- raphy (HPLC) has been shown to be a powerful tool for use in the purification of fatty acid oxidation products. For example, the lipid hydroperoxide products formed from lipoxy- genase reactions of arachidonic acid (7,8) have been prepared by air oxidation of the fatty acid and purified by HPLC (9,10). Further, the first isolable products in prostaglandin bio- synthesis, PGG 2 and PGH2, (11,12) have recently been purified by HPLC (13). PGG~ and PGH 2 are both peroxide products of limited stability as are the lipoxygenase pro- ducts 12-hydroperoxy and 5-hydroperoxy arachidonic acid. The fact that these com- pounds may be purified by HPLC suggested to us that HPLC might also be used in the study of phospholipid oxidation. We recently reported (14) that different lecithin molecular species could be separated by reverse phase HPLC and the primary compo- nents of egg lecithin could also be purified by this technique. Further, it was noted that after storage at low temperature for several months, 1-palmitic-2-1inoleic-phosphatidylcholine was converted to a new product. This new product was readily separated from the unreacted lecithin by reverse phase HPLC and we sus- pected it to be an oxidation product of 1-pal- mitic-2-1inoleic-phosphatidylcholine. We report here on the isolation and characterization of lecithin air oxidation products. HPLC may be used to purify the primary lecithin oxidation products and we note that these primary oxidation products are lecithin hydroperoxides. MATERIALS AND METHODS Fatty acids were obtained from NuChek Prep (Elysian, MN) and used without further purification. Egg lecithin was obtained from Sigma Biochemical Co. (St. Louis, MO). Lecithin Synthesis l-Palmitic-2-1inoleic-phosphatidylcholine (1P, 2L-PC) was prepared by the method reported by Gupta et al. (15). Thus, egg lecithin was converted to glycerophosphorylcholine which was then diacylated to give dipalmitic phosphatidylcholine (di P-PC). Snake venom hydrolysis followed by reacylation gave 1P, 2L-PC. The product was characterized by 1H and t3C nuclear magnetic resonance (NMR) spectroscopy and was pure by thin-layer chromatography (TLC) and HPLC. 1-Stearic-2- arachidonic phosphatidylcholine was prepared by a similar approach (15). I63