Rate of Lipid Peroxyl Radical Production during Cellular Homeostasis Unraveled via Fluorescence Imaging

Reactive oxygen species (ROS) and their associated byproducts have been traditionally associated with a range of pathologies. It is now believed, however, that at basal levels these molecules also have a beneficial cellular function in the form of cell signaling and redox regulation. Critical to elucidating their physiological role is the opportunity to visualize and quantify the production of ROS with spatiotemporal accuracy. Armed with a newly developed, extremely sensitive fluorogenic alpha-tocopherol analogue (H4BPMHC), we report herein the observation of steady concentrations of lipid peroxyl radicals produced in live cell imaging conditions. Imaging studies with H4BPMHC indicate that the rate of production of lipid peroxyl radicals in HeLa cells under basal conditions is 33 nM/h within the cell. Our work further provides indisputable evidence on the antioxidant role of Vitamin E, as lipid peroxidation was suppressed in HeLa cells both under basal conditions and in the presence of Haber-Weiss chemistry, generated by the presence of cumyl hydroperoxide and Cu2+ in solution, when supplemented with the alpha-tocopherol surrogate, PMHC (2,2,5,7,8-pentamethyl-6-hydroxy-chromanol, an alpha-tocopherol analogue lacking the phytyl tail). H4BPMHC has the sensitivity needed to detect trace changes in oxidative status within the lipid membrane, underscoring the opportunity to illuminate the physiological relevance of lipid peroxyl radical production during cell homeostasis and disease.