Characterization of Retinal Pigment Epithelial Melanin and Degraded Synthetic Melanin Using Mass Spectrometry and In Vitro Biochemical Diagnostics

Abstract

With increasing age, there is an observable loss of melanin in retinal pigment epithelial (RPE) cells. It is possible that degradation of the pigment contributes to the pathogenesis of retinal disease, as the cellular antioxidant material is depleted. Functionally, intact melanin maintains protective qualities, while oxidative degradation of melanin promotes reactive oxygen species (ROS) generation and formation of metabolic byproducts, such as melanolipofuscin. Understanding the structural and functional changes to RPE melanin with increasing age may contribute to a better understanding of disease progression and risk factors for conditions such as age-related macular degeneration (AMD). In this study, human donor RPE melanin is characterized using MALDI mass spectrometry to follow melanin degradation trends. In vitro models using ARPE-19 cells are used to assess photo-reactivity in repigmented cells. Significant protection against intracellular ROS produced by blue light is observed in calf melanin-pigmented cells versus unpigmented and black latex bead controls (P < 0.0001). UV-B exposure to aged human melanin-pigmented cells results in a significant increase in nitric oxide production versus control cells (P < 0.001). Peroxide-treated synthetic melanin is characterized to elucidate degradation products that may contribute to RPE cell damage.