14] Peroxidation of phosphatidylserine in mechanisms of apoptotic signaling

Abstract

Oxidative stress is a frequent trigger of apoptosis in a variety of cells and is also thought to be involved as a component of the common pathway in the execution of apoptosis. While effects of oxidative stress on protein components of apoptotic machinery, such as caspases, have been well characterized, information on selective oxidation of specific classes of phospholipids in live cells is limited. The direct measurement of oxidized lipid products is problematic, as cells possess a very effective system for remodeling and repairing oxidatively modified phospholipids that interferes with their accurate measurement. The authors have developed a sensitive, specific, and reliable procedure for the assessment of oxidative stress in different classes of membrane phospholipids in intact live cells based on their metabolic acylation with an oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid (cis-PnA), as a reporter molecule. Basic procedures and some applications of the technique are described in the chapter. The model of cis-PnA-labeled cells offers a unique model system for quantitative studies of oxidative stress and selective oxidation of specific classes of phospholipids under normal physiological conditions, as well as during cell injury and apoptotic death.