Consecutive action of phospholipase A2 and glutathione peroxidase is required for reduction of phospholipid hydroperoxides and provides a convenient method to determine peroxide values in membranes

Abstract

The purpose of this study was to investigate the ability of selenium-dependent glutathione peroxidase to reduce phospholipid hydroperoxides in membrane bilayers and to develop a method to measure the peroxide content of phospholipids. Phospholipid hydroperoxides were synthesized by photooxidation of 1-palmitoyl 2-linoleoyl phosphatidylcholine and characterized by gas chromatography-mass spectrometry. Phospholipid hydroperoxides in phosphatidylcholine bilayers showed no detectable reactivity with Se-dependent glutathione peroxidase (the reaction is at least 65,000 times slower than with an available hydroperoxide). However, after the phospholipid hydroperoxides were preincubated with phospholipase A2, the free fatty acid hydroperoxides became available as a substrate for Se-dependent glutathione peroxidase. The enzyme assay can be used for convenient determination of peroxide values in phospholipids at the 1 nmole level and free fatty acid hydroperoxides can be distinguised from phospholipid hydroperoxides by omitting phospholipase A2. The accuracy of the enzymatic method was confirmed using an improved colorimetric chemical assay to measure peroxide values of phospholipid hydroperoxides to the same sensitivity. The chemical assay was not linear in the presence of high levels of lipid, but at low levels of lipid the peroxide values of phospholipid hydroperoxides measured by both methods agreed to within 1%. Since high levels of lipid inhibited the chemical assay, the enzyme assay is more accurate for determination of peroxides in membranes and tissues. The possible role of phospholipase deficiencies as a causal factor in degenerative diseases thought to be due to lipid peroxidation, such as Neuronal Ceroid Lipofuscinosis (Battens disease), is discussed.