Abdel-Rahman 2015 Abstract MiPschool Cape Town 2015

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Assessment of NADPH-dependent oxygen consumption and hydrogen peroxide production by high-resolution respirometry and EPR spectroscopy.


Abdel-Rahman EA, Khalifa ARM, Mahmoud AM, Ali MH, Ali SS (2015)

Event: MiPschool Cape Town 2015

Assessment of NADPH-dependent oxygen consumption and hydrogen peroxide production by High-Resolution Respirometry and EPR spectroscopy. Centre Of Aging And Associated Diseases, Helmy Institute Of Medicine, Zewail City Of Science and Technology, Giza, Egypt, 12588. 2Dep Of Pharmacology, School Of Medicine, Assiut Univ, Assiut, Egypt, 71515. NADPH oxidase (Nox) is emerging as one of the major sources of cellular reactive oxygen species (ROS). While controlled ROS generation by Nox is involved in the redox regulation of physiological cellular processes, excessive ROS production leads to tissue damage (Ali et al., 2010). Nox over-reactivity has been shown to mediate the pathogenesis of tissue injury in neurodegenerative disorders (Dugan et al., 2009), ischemia-reperfusion and cardiovascular disorders. Because of the short-lived nature of ROS, it is challenging to assess and monitor ROS levels in biological specimens. Thus, the development of a method to measure NADPH oxidase-derived ROS generation would be a valuable research tool to understand mechanisms relevant to neurodegeneration and tissue injury. Furthermore, this approach might be of relevance for screening of novel Nox inhibitors, which may selectively reduce disease-related Nox-mediated ROS generation without modifying ROS physiological signaling function. By using the Oroboros O2k Station, we applied two different protocols for measuring oxygen consumption in parallel with ROS levels in freshly isolated synaptosomes. In parallel with spin trapping EPR spectroscopy, we employed this protocol to delineate the contribution of NADPH oxidase to ROS production in young female and male C57BL6 mice. The first protocol based on using a polarographic high resolution O2k sensor to measure oxygen consumption and a fluorescence-based module to monitor the rate of NADPH-mediated hydrogen peroxide production. Consistent Nox-dependent oxygen consumption was detected in synaptosomes following activation of Nox by 5 mM NADPH (3 doses). In parallel, we also employed a WPI -electrochemical sensor to determine H2O2 in the same sample. Although we didn't detect sex-dependent discrepancy in the rate of hydrogen peroxide production by Nox in isolated synaptosomes, the HRP/Amplex Red system was associated with greater oxygen consumption and higher rates of hydrogen peroxide generation, suggesting that HRP may be inducing Nox-like activity. We verified the Nox activity using spin trapping EPR spectroscopy. Our study revealed that the OROBOROS O2k can be successfully used for assessment of Nox activity through the parallel detection of oxygen consumption and the resulting hydrogen peroxide generation. However, we have also found that HRP exhibit NADPH-dependent, oxygen-consuming, and H2O2 -producing activity. Efforts are currently exerted to test other redox-sensitive dyes for the detection of ROS in the absence of HRP.

O2k-Network Lab: ET Giza Ali SS