An assay for the rate of removal of extracellular hydrogen peroxide by cells☆

Cells have a wide range of capacities to remove extracellular hydrogen peroxide. At higher concentrations of extracellular H2O2 (micromolar) the rate of removal can be approximated by a rate equation that is first-order in the concentration of H2O2 and cell density. Here we present a method to determine the observed rate constant for the removal of extracellular H2O2 on a per cell basis. In the cells examined, when exposed to 20 μM H2O2, these rate constants (kcell) range from 0.46 × 10-12 s-1 cell-1 L for Mia PaCa-2 cells (human pancreatic carcinoma) to 10.4 × 10-12 s-1 cell-1 L for U937 cells (human histiocytic lymphoma). For the relatively small red blood cell kcell = 2.9 × 10-12 s-1 cell-1 L. These rate constants, kcell, can be used to compare the capacity of cells to remove higher levels of extracellular H2O2, as often presented in cell culture experiments. They also provide a means to estimate the rate of removal of extracellular H2O2, rate = - kcell [H2O2] (cells L-1), and the half-life of a bolus of H2O2. This information is essential to optimize experimental design and interpret data from experiments that expose cells to extracellular H2O2.