The purpose of this study is to develop a higher sensitivity method to detect phospholipid peroxides in tissues to more effectively investigate the role of lipid peroxidation in pathology. We recently presented a gas chromatography-mass spectrometry (GC-MS) method for identification and measurement of phospholipid peroxides at the 10 ng level (van Kuijk, et al. J. Free Rad. Biol. Med., 1: 215-225, 1985) based on transesterification to form fatty acid methyl ester derivatives. While this method is quite usefule under some circumstances, more sensitivity was desirable to analyse trace amounts of lipid peroxidation products in small samples of various tissues. We present a method to prepare pentalfluorobenzyl esters of fatty acids and hydroxy fatty acids directly from phospholipids by transesterification at room temperature or from triglycerides at 60%C. Oxidized lipids are extracted into dichloromethane and peroxide functions are reduced with sodium borohydride. Transesterification of phospholipids is carried out in dichloromethane with 20% (v/v) pentafluorobenzyl alcohol, 1% (w/v) potassium tertbutoxide and 0.2 N (m-trifluoromethyl phenyl) trimethyl ammonium hydroxide at room temperature for 30 min. Pentafluorobenzyl esters are purified on open silica columns. O-trimethyl silyl derivatives of the alcohol functions are formed, and GC-MS is carried out with negative ion chemical ionization detection. The single or double oxidation products of oleate, linoleate, arachidonate, and docosahexaenoate were detected with 10 pg sensitivity in oxidized rat retina phospholipids or synthetic phospholipids by negative ion chemical ionization with specific ion monitoring of carboxylate anions.
Bibliographical noteFunding Information:
Acknowledgments--This work was supported by the Netherlands Organization for the Advancement of Pure Research (ZWO) (to FJGMvK), and by grants from the U.S. National Eye Institute (to EAD and RJS). We thank Kenton Parker and Dr. Alan Deese for samples of ( 16: 0)(22: 6) phosphatidylcholine and ( 16: 0)(d 12-22 : 6) phosphatidylcholine. We thank Dr. J. Konopelski and R. M. Par-khurst for useful discussions.
- Gas chromatography-mass spectrometry
- Lipid peroxidation
- Pentafluorobenzyl esters
- Phospholipid hydoperoxides
- Polyunsaturated fatty acid hydroperoxides