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Respiration state IV-generated ROS destroy the mitochondrial bilayer packing order in vitro. An EPR study
Francesco M Megli1, Karen Sabatini
1Dipartimento di Biochimica e Biologia Molecolare, Università di Bari, CNR, Via E. Orabona 4, 70126 Bari, Italy. f.m.megli@biologia.uniba.it
Abstract:
The aim of the present study was to detect defective structural properties in bilayers of mitochondrial phospholipids after oxidative stress of isolated mitochondria in vitro, reportedly during respiration state IV. The structural behaviour of extracted phospholipids was studied by electron paramagnetic resonance (EPR) spectrometry in oriented phospholipid bilayers spin-labelled with 5-doxyl-lecithin, by detecting of the degree of EPR spectral anisotropy loss, indicative of the phospholipid bilayer packing order. Bilayers of phospholipids from untreated mitochondria showed the highest spectral anisotropy, hence highly ordered structure, while chemically oxidised phospholipid yielded almost completely disordered supported phospholipid bilayers. Samples from mitochondria after respiration state IV showed bilayer disorder increasing with oxidation time, while inclusion of the antioxidant resveratrol in the respiration medium almost completely prevented bilayer disordering. On the other hand, beta-n-doxylstearoyl-lecithin spin-labelled mitochondria showed unchanged order parameter S at C positions 5, 12 and 16 after respiration state IV, confirming the insensitivity of this parameter to phospholipid oxidative stress. It is concluded that reactive oxygen species attack to the membrane affects lipid packing order more than fluidity, and that EPR anisotropy loss reveals oxidative damage to the bilayer better than the order parameter.
Insights
Oxidative stress damages mitochondrial phospholipid bilayers, decreasing their structural order. The antioxidant resveratrol protected against this damage, indicating EPR anisotropy loss is a sensitive indicator of oxidative stress.
Area of Science:
- Mitochondrial biochemistry
- Membrane biophysics
- Oxidative stress research
Background:
- Mitochondria are crucial for cellular energy production.
- Oxidative stress can impair mitochondrial function.
- Phospholipid bilayer structure is vital for membrane integrity.
Purpose of the Study:
- To investigate structural changes in mitochondrial phospholipid bilayers under oxidative stress.
- To assess the impact of respiration state IV on mitochondrial membrane structure.
- To evaluate the protective effect of resveratrol against oxidative damage.
Main Methods:
- Isolated mitochondria subjected to in vitro oxidative stress.
- Electron Paramagnetic Resonance (EPR) spectrometry using spin-labeled phospholipids.
- Analysis of EPR spectral anisotropy loss to determine bilayer packing order.
Main Results:
- Oxidative stress significantly reduced phospholipid bilayer order.
- Respiration state IV induced progressive bilayer disordering.
- Resveratrol treatment largely prevented the loss of bilayer order.
- Order parameter S at specific carbon positions was insensitive to oxidative stress.
Conclusions:
- Reactive oxygen species primarily affect lipid packing order over fluidity.
- EPR anisotropy loss is a more sensitive marker of oxidative damage than the order parameter.
- Resveratrol demonstrates protective effects against mitochondrial oxidative damage.