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 beta-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.