Simultaneous measurements of the intra- and extra-cellular oxygen concentration in viable cells

An EPR method that can measure the intra- and extra-cellular oxygen concentration [O2] simultaneously in vitro has been developed using specially designed nitroxides. In the presence of Fe(CN)6(3-) in the medium, intracellular [O2] is measured by a neutral 15N-nitroxide and extracellular [O2] is measured by a negatively charged 14N-nitroxide, since charged species do not enter cells and the EPR spectrum of a 15N-nitroxide does not overlap with that of a 14N-nitroxide. The method is based in part on the minimal broadening of negatively charged nitroxides by Fe(CN)6(3-) and the very effective broadening of neutral nitroxides by the same paramagnetic ions. Results with this method confirm the existence of gradients in [O2] between the extracellular and intracellular compartments in CHO cells and M5076 tumor cells, even without stimulation of cellular respiration by CCCP. The nature of the barrier that needs to be involved to account for the experimental results raises some significant questions.