Microximetry: Simultaneous Determination of Oxygen Consumption and Free Radical Production Using Electron Paramagnetic Resonance Spectroscopy

The results presented in this article demonstrate that EPR oximetry based on the LiPc microcrystalline powder as a probe in combination with the DEPMPO spin trap can be used for the simultaneous measurement of oxygen consumption and reactive oxygen-free radical species in enzymatic reactions and cellular respirations. Because the sample requirement is less than 20 μl, the present technique is ideally suited for cases where very little sample is available. From the illustrated examples, it is clear that such an experiment is not restricted only to enzymatic reactions, but can also be applied for cellular respirations. Especially in the lower oxygen concentration range, the EPR oximetry method is very accurate, unlike the established methods. The DEPMPO used in the present work is also a reasonably good spin trap with a relatively higher spin adduct half-life time of the spin adduct. The quantitative analysis of the concentration is a little complex, as there is also simultaneous decomposition. However, with appropriate computations, the absolute concentration of superoxide and OH radicals could be determined. From the results obtained in these studies, it is estimated that about 30% of the consumed oxygen is leaked as free radicals. Although we have illustrated simple uses of this technique as examples, this technique can be used to study the effects of specifically blocked or induced individual steps on the overall oxygen consumption and superoxide productions in different cell lines.