Cholesterol as a natural probe for free radical-mediated lipid peroxidation in biological membranes and lipoproteins

Abstract

We describe a relatively convenient and reliable procedure for assessing the magnitude of free radical-mediated (chain) lipid peroxidation in biological systems. The approach is based on use of radiolabeled cholesterol ([14C]Ch) as a probe and determination of well-resolved oxidation intermediates/products ([14C]ChOX species), using high performance thin layer chromatography with phorphorimaging detection (HPTLC-PI). In a lipid hydroperoxide-primed liposomal test system treated with ascorbate and a lipophilic iron chelate, the following well-resolved [14C]ChOX are detected and quantified: 7α/7β-OOH, 7α/7β-OH, and 5,6-epoxide, their levels increasing with incubation time at 37 °C. [14C]Ch also serves as an excellent probe for lipid peroxidation in lipoproteins and plasma membranes of mammalian cells. Because this approach utilizes Ch as a natural in situ probe, it eliminates potential artifacts associated with artificial probes such as spin traps and fluorophores.

Dyslipidemia and non-small cell lung cancer risk in Chinese population: a case-control study

“…Cholesterol, one of the components of the membrane, plays a crucial role in maintaining the integrity of the cell membrane [ 22 ]. In addition, cholesterol was reported to accumulate in specific domains of the membrane and combine with sphingolipids, which formed compartmentalized and highly stable micro-domains termed lipid-rafts [ 23 ].…”

Cholesterol Hydroperoxide Generation, Translocation, and Reductive Turnover in Biological Systems

“…A great improvement came with the authors’ introduction of two highly sensitive and specific new approaches: (i) high performance liquid chromatography with mercury cathode electrochemical detection (HPLC-EC(Hg)) for analyzing individual ChOOHs [14–16] and (ii) high performance thin layer chromatography with phosphorimaging detection (HPTLC-PI) for analyzing [ 14 C]ChOX [17,18]. Using HPLC-EC(Hg), we found that tracking growth of the 7α/7β-OOH signal and decline of the initial 5α-OOH signal provided an excellent means of assessing LOOH-initiated chain peroxidation in cells photodynamically stressed with 1 O 2 [19].…”

Cholesterol Peroxidation as a Special Type of Lipid Oxidation in Photodynamic Systems

“…Transfer rate can be increased using a protein transporter such as recombinant sterol carrier protein‐2 (SCP‐2), which is easily removed sufficient transfer uptake is achieved. For mammalian cells, most of the [ 14 C]Ch transferred in this way will reside in the plasma membrane, allowing chain peroxidation activity to be probed specifically in this compartment . Another plus about using [ 14 C]Ch is that it avoids the potential artifacts associated with exogenous probes such as spin traps and fluorophores , which might perturb lipid organization or act as antioxidants in systems being studied.…”

“…During chain reactions induced by an oxidative challenge in the presence of reducible metal ions or reductive turnover of pre‐existing LOOHs, the probe gives rise to [ 14 C]ChOX species. The latter are conveniently detected and quantified by high‐performance thin layer chromatography with phosphorimaging detection (HPTLC‐PI) . Individual ChOX species include redox‐active 7 α / β ‐OOH as well as redox‐inactive 7 α / β ‐OH, 7‐ketone, and 5,6‐epoxide.…”

“…signals are obtained directly without having to solubilize and extract lipids. Nevertheless, the [ 14 C]Ch approach is highly sensitive and straightforward in application and has been successfully applied to a number of reaction systems, ranging from liposomes and lipoproteins to membranes of living cells .…”