Methodology for measuring oxidative capacity of isolated peroxisomes in the Seahorse assay.

Abstract

The regulation of cellular energetics is a complex process that requires the coordinated function of multiple organelles. Historically, studies focused on understanding cellular energy utilization and production have been overwhelmingly concentrated on the mitochondria. While mitochondria account for the majority of intracellular energy production, they alone are incapable of maintaining the variable energetic demands of the cell. The peroxisome has recently emerged as a secondary metabolic organelle that complements and improves mitochondrial performance. Although mitochondria and peroxisomes are structurally distinct organelles, they share key functional similarities that allows for the potential to repurpose readily available tools initially developed for mitochondrial assessment to interrogate peroxisomal metabolic function in a novel manner. To this end, we report here on procedures for the isolation, purification and real-time metabolic assessment of peroxisomal β-oxidation using the Agilent Seahorse® system. When used together, these protocols provide a straightforward, reproducible and highly quantifiable method for measuring the contributions of peroxisomes to cellular and organismal metabolism.