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

Abstract

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.

4-hydroxynonenal causes impairment of human subcutaneous adipogenesis and induction of adipocyte insulin resistance

“…The arbitrary units, relative fluorescence units (RFU), were based directly on fluorescence intensity. Intracellular ROS production was also evaluated using aminotriazole assay as described previously [37] using OxiSelect hydrogen peroxide/peroxidase assay kit following 12 hours of 10µM 4-HNE treatment according to manufacturer's instructions.…”

Localized redox relays as a privileged mode of cytoplasmic hydrogen peroxide signaling

“…(1) we model the diffusion of the peroxiredoxin forms and of the H 2 O 2 according to Fick's laws [49]. Per-cell H 2 O 2 removal rate constants determined by Wagner et al [50] allow to estimate the permeability constants in the range 10 − 10 m s…”

Frequency modulated translocational oscillations of Nrf2, a transcription factor functioning like a wireless sensor

“…Our recent pilot studies suggest Nrf2 oscillation responds to a single bolus exposure of 10-20 μM H 2 O 2 in cultured cells (Xue, M., Rabbani N. and Thornalley, P.J., unpublished observation); approximately 400 μM H 2 O 2 required to stabilize Nrf2 protein [27]. Given the high rate of metabolism of exogenous H 2 O 2 [28], the lower bolus exposure may induce responses similar to those of endogenous oxidative challenge to homoeostasis.…”