Intermembrane translocation of lipid hydroperoxides: Transfer kinetics and prooxidant effects

Oxidative pressure in cells may predispose unsaturated membrane lipids to oxidative modification, e.g. formation of phospholipid (PL)- and cholesterol (Ch)-derived hydroperoxides (PLOOHs, ChOOHs). Lipid hydroperoxides (LOOHs) may be generated by chain peroxidation reactions triggered by iron-catalyzed formation of hydroxyl, alkoxyl, or peroxyl radicals. Another source of LOOHs is photodynamic action, i.e. light-induced oxidation mediated by a sensitizing agent such as a flavin or tetrapyrrole. Singlet molecular oxygen ( 1O₂), a common intermediate in photodynamic reactions, can form LOOHs by adding directly to unsaturated PLs or Ch. Once formed in cell membranes, LOOHs can undergo (i) one-electron reduction, thus triggering free radical chain peroxidation (toxicity enhancement) or (ii) enzymatic two electron reduction to relatively innocuous alcohols (toxicity containment). Alternatively, LOOHs might translocate to other membranes before undergoing such reactions. Being more polar than parent lipids, LOOHs would desorb more readily from donor membranes and this would favor transfer. The central hypothesis of this research is that LOOH translocation provides a mechanism for broadcasting peroxidative stress, both within and between cells. This might explain how plasma membrane peroxidative damage, for example, might signal mitochondria to initiate an apoptotic cascade.; The aim of this project has been to test the above hypothesis, using high-performance LOOH analytical techniques such as liquid chromatography with reductive electrochemical detection [HPLC-EC(Hg)] or thin layer chromatography with phosphorimaging radiodetection (HPTLC-PI). In initial studies, spontaneous transfer kinetics of LOOHs in model systems were examined, e.g. photoperoxidized erythrocyte ghost donors and small liposome (SLV) acceptors. First-order transfer rate constants for various HPLC-EC(Hg)-detectable ChOOH species relative to parent Ch were found to decrease in the following order: 7αβ-OOH > 5α-OOH > 6α-OOH > 6β-OOH >> Ch. Also we examined the rate constants for various PLOOH families relative to parent PLs were ranked as follows: PCOOH ≅ PEOOH ≅ PSOOH > SMOOH. Protein-facilitated LOOH translocation has also been investigated, using low-specificity sterol carrier protein-2 (SCP-2) as a mediator. SCP-2 is known to bind and transfer a variety of lipid species (including sterols, PLs, and fatty acyl-CoAs) and plays a role in their metabolism and homeostasis. An additional hypothesis in this project is that SCP-2 mediates intracellular LOOH trafficking, which either attenuates or enhances LOOH toxicity. Kinetic studies with purified bovine liver SCP-2 and with human recombinant SCP-2 have shown that this protein greatly accelerates ChOOH and PLOOH translocation over spontaneous backgrounds. This is at least partially explained by LOOH pick-up at the donor membrane and delivery through the aqueous compartment as an SCP-2-LOOH complex.; These are the first studies to demonstrate spontaneous and protein-facilitated intermembrane transfer of ChOOHs and PLOOHs. They describe a previously unrecognized mechanism by which peroxidative stress might be disseminated.