Phospholipid bilayers are the main component of the cellular membrane, which is a theater for many biological reactions. The addition of peroxide groups to lipids, lipid peroxidation, is a ubiquitous phenomenon related to  aging and a variety of diseases. Specifically, a recently discovered cell death known as ferroptosis has been found to be characterized by the accumulation of lipid peroxides in cells. The dominant lipid peroxidation pathway in ferroptosis, whether it is through enzymatic or non-enzymatic reactions, is unknown and the subject of controversy. The goals of the current study are to determine the dominant lipid peroxidation pathway in bilayers and investigate how lipid peroxidation ultimately leads to cellular death by establishing a quantitative measurement of lipid peroxidation in model membranes. To determine the dominant lipid peroxidation pathway, the fluorescent probe diphenylhexatriene (DPH) was incorporated into membrane systems such as small unilamellar vesicles (SUVs) and supported lipid bilayers (SLBs). DPH has been previously reported to lose fluorescence upon oxidation by lipid peroxides, and we have shown that it is sensitive to different lipid peroxide compositions in SUVs and SLBs. SUVs were used to relate DPH fluorescence to formation of lipid peroxides, observed using absorbance spectroscopy. The reaction was also reconstituted in SLBs to investigate the effects lipid peroxides have on bilayers and its non-lipid components by using fluorescence microscopy techniques such as total internal reflection fluorescence (TIRF) and fluorescence correlation spectroscopy (FCS).

Fluorescence investigation of lipid peroxidation in in-vitro phospholipid bilayer membranes

ACS Spring 2022

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