Sensitive and real-time determination of H2O2 release from intact peroxisomes

Peroxisomes are essential and ubiquitous cell organelles having a key role in mammalian lipid and oxygen metabolism. The presence of flavine oxidases makes them an important intracellular source of H₂O₂: an obligate product of peroxisomal redox reactions and a key reactive oxygen species. Peroxisomes proliferate in response to external signals triggered by peroxisome-proliferator-activated receptor signalling pathways. Peroxisome-derived oxidative stress as a consequence of this proliferation is increasingly recognized to participate in pathologies ranging from carcinogenesis in rodents to alcoholic and non-alcoholic steatosis hepatitis in humans. To date, no sensitive approach exists to record H₂O₂ turnover of peroxisomes in real time. Here, we introduce a sensitive chemiluminescence method that allows the monitoring of H₂O₂ generation and degradation in real time in suspensions of intact peroxisomes. Importantly, removal, as well as release of, H₂O₂ can be assessed at nanomolar, non-toxic concentrations in the same sample. Owing to the kinetic properties of catalase and oxidases, H₂O₂ forms fast steady-state concentrations in the presence of various peroxisomal substrates. Substrate screening suggests that urate, glycolate and activated fatty acids are the most important sources for H₂O₂ in rodents. Kinetic studies imply further that peroxisomes contribute significantly to the β-oxidation of medium-chain fatty acids, in addition to their essential role in the breakdown of long and very long ones. These observations establish a direct quantitative release of H₂O₂ from intact peroxisomes. The experimental approach offers new possibilities for functionally studying H₂O₂ metabolism, substrate transport and turnover in peroxisomes of eukaryotic cells.