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.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► We present a method to determine a rate constant, kcell, for removal of extracellular H2O2 by cells. ► There is a wide range of capacity of cells to remove extracellular H2O2. ► Red blood cells have a high capacity to remove extracellular H2O2, despite their small size. ► kcell is an invaluable tool to guide experimental design and inform data interpretation.