A fluorescence plate reader assay for monitoring the susceptibility of biological samples to lipid peroxidation

The susceptibility of biological samples to lipid peroxidation can be determined by exposing samples to a lipid peroxidation initiator and measuring the length of time prior to the onset of lipid peroxidation. Previous studies have shown that aldehydes generated by lipid peroxidation can react with amines to produce fluorescent products. We have utilized this principle to develop a fluorescence plate reader assay for measuring susceptibility to lipid peroxidation. In this assay, samples are placed in glycine/phosphate buffer and loaded into a 96-well plate. Lipid peroxidation initiators are added, and fluorescence is monitored over time. Samples were assayed for susceptibility to lipid peroxidation by both the thiobarbituric acid reactive substances assay and the fluorescence plate reader assay. We found good agreement between these two methods in assessing relative susceptibility to lipid peroxidation in liver microsomes and mitochondria. The fluorescence assay was also used to monitor lipid peroxidation in liposomes and rat liver homogenates. Fluorescence was stable over an extended time period and could be induced by a variety of lipid peroxidation initiators. The fluorescence plate reader assay offers a rapid method for monitoring lipid peroxidation in a large number of samples.